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Introduction
Gibberellins (GAs) are plant growth hormones that regulate various processes, including stem elongation, seed germination, dormancy, flowering, sex expression, enzyme induction, and fruit senescence [1]. Gibberellic acid (GA3) is a subtype of GAs that, when accumulated in the soil for months and consumed by various exposed vegetables and fruits, can cause toxicity and have hazardous effects on the health of humans and animals [2]. GA3 has been shown to modify antioxidative systems induced by free radical production in various organs [3], tissue carcinogenic, persuade tumor formation [4], affect sexual differentiation and mammal fertility [5].
The development of the ovary depends on the development and maturation of ovarian follicles; a process of folliculogenesis [6]. The development of ovaries in rats starts before birth, with the primary change being the formation of follicles [7]. During the initial stages of ovarian development, the ovalocytes are arrested in the meiosis-I phase while awaiting two essential developmental processes: (1) formation of the primordial follicle, and (2) transition from primordial to the primary follicle. This process takes in (a) embryonic days from 18 to 21 (E18–21) as the majority of the oocytes in the rats were in their places and the apoptosis was observed, (b) the peak days from postnatal day (PND) 0 to 3 in which the apoptosis and primordial follicle formation is present, and (c) the final stage from PND 4 to 7; where all oocyte cavities have been demolished, resulting in no observed oocytes in the ovaries, and a transition from primordial to primary follicles is present in the most of the ovarian cortex [8]. In the neonatal period (PND 0–7); the ovarian follicle development is independent of the pituitary gonadotropins luteinizing hormone (LH) or follicle stimulating hormone (FSH), and the follicles remain in the preantral stage [9]. The peripubertal period in rats lasts from (PND 33–37), and pubertal periods from (PND 38–46) which depends mainly on pituitary gonadotropins (LH and FSH) [10]. Postnatal ovarian development, or folliculogenesis, occurs when the primordial follicle pool matures into primary, then secondary follicles independently of gonadotropins. However, this process is closely regulated by both activating and inhibitory factors making it more suspectable to internal or external environmental toxicity. The development of a secondary follicle into a Graafian follicle is completely dependent on gonadotropins, with ovulation relying on this process [11].
Selenium (Se) is an essential micronutrient required for the function of various Se-dependent enzymes, known as selenoprotiens, which are crucial for every animal cell [12]. Se has been shown to have antioxidant properties that help protect cells from oxidative damage [13]. To our knowledge, this is the first study assigned to assess the effect of GA3 toxicity on pre and postnatal ovarian development and to investigate the expected protective effect of Se against GA3-induced embryotoxicity during pre and postnatal stages of ovarian development in albino rats.
Materials and Methods
Chemicals
GA3: Berelex tablets (10 g) contain GA3 10% tablet manufactured (Valent BioSciences).
The stock solution for GA3: was prepared in saline and refreshed as needed during the experiment.
Sodium selenite powder (Na2SeO3; Sigma-Aldrich) was dissolved in distilled water.
Animals’ husbandry and experimental design
This study was conducted at the animal house of the Faculty of Medicine, Suez Canal University. According to a statistical sample size equation [14]; forty-eight females with an equal number of male rats obtained from the animal house of Suez Canal University were used in this study. Animals were adapted to laboratory conditions with normal light and temperature, having free access to water and food. The current experiment was conducted after obtaining the approval permission of the Ethics Committee at the Faculty of Medicine, Suez Canal University (5132#). All experiments in this study occurred according to the laboratory animal care guidelines, guaranteeing the study followed Good Laboratory Practice procedures. After one week of acclimatization, two experimental procedures were done on two different age groups in this study. The first experiment: was done on adult mature female rats weighting (200–250 g) that were prepared for mating (each exposed female was mated with one mature male 1:1). After making sure of pregnancy by finding sperms in their vagina was considered the first day of pregnancy (GD1), then the pregnant rats were divided into 3 groups. The second experiment: was done on prepubertal female albino rats (4 weeks old) with an average body weight of 50–60 g. The rats in the two experiments (n=24 for each) were divided into 3 groups for each group.
Animal grouping
Group I: served as control (n=12) and was divided into 2 subgroups as follows:
Group Ia; (negative control): the rats received distilled water (0.01 ml/g body weight) without any treatments.
Group Ib; (positive control): the rats received sodium selenite in a dose of 0.3 mg/kg/body weight [15].
Group II (GA3 treated, n=6): rats received GA3 (55 mg/kg equivalent to 1/100 of lethal dose 50) via gastric gavage [16].
Group III (Se-protected, n=6): rats received GA3 by the previous dose plus sodium selenite in a dose of 0.3 mg/kg/body weight via gastric gavage.
The treatments in the first experiment began at the gestational day 7 [6] until day four postnatal (PND 4) and 2 weeks in the pubertal rats in the second experiment. The ovaries of the rat’s offspring in the 1st experiment and from the pubertal rats in the 2nd experiment were taken for different histological and immunohistochemical analyses.
Ovarian oxidative stress marker evaluation
For assessing oxidative stress marker levels; samples of the ovarian tissues from all groups were frozen in liquid nitrogen, then homogenized in potassium phosphate buffer solution (50 mM, pH 7.5) using a Potter-Elvehiem homogenizer to give a 10% homogenate. Homogenates were centrifuged at 1,500 g for 10 minutes at 4°C. The supernatant was collected, placed on ice, and immediately used to determine malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT). MDA, a marker for lipid peroxidation, was measured calorimetrically in ovarian homogenate according to the specified method by Ohkawa et al. [19]. SOD is a group of antioxidant enzymes that regulate reactive oxygen species (ROS) levels by catalyzing the conversion of superoxide into hydrogen peroxide and molecular oxygen. The activity of SOD was determined in ovarian tissues according to the method of Nishikimi et al. [20]. The principle of this method is based on the ability of SOD to inhibit the reduction power of phenazine methosulphate, which mediates the reduction of nitro blue tetrazolium. CAT is an antioxidant enzyme that degrades hydrogen peroxide into water and oxygen. The CAT activity was assayed using a spectrophotometric assay developed by Góth [21], which measures the stable complex formation of hydrogen peroxide with ammonium molybdate at an absorbance of 405 nm.
Histological and immune-histochemical examination
Samples of ovarian tissues from the first experiment (neonatal rats; PND 4) and the second experiment (pubertal rats) from all studied groups were fixed in Carnoy’s fixative. The fixed ovarian tissues were dried using ultimate ethanol, cleaned with methyl benzoate, and later infiltrated with paraffin wax. The studied paraffin blocks of the ovarian tissues were cut into 5 μm thick sections using a microtome. The ovarian sections were stained with hematoxylin and eosin (H&E) for general histopathological findings and with a Masson Trichrome stain to detect collagen deposition and fibrosis.
Immunohistochemical staining was done using Ki-67 antibodies to assess the proliferative activity marker of the developing follicles and androgen receptors (ARs) antibodies expression to evaluate the degree of maturation of hormonal receptors and their responses to androgens of the ovarian follicles. The slides were dried after being mounted to eliminate any water trapped underneath them. The slides were washed with tris-buffered saline buffer after being immersed in distilled water. Endogenous peroxidase was inhibited with hydrogen peroxide for 10 minutes, rinsed with buffer for 1 minute, and then digested with proteinase K, followed by a 2-minute rinse. The protein block was added for 10 minutes before being shaken off. Anti-Ki-67 [22], and primary ARs antibodies [23] were applied to each tissue and cleaned with buffer for 2 minutes, followed by an antibody enhancer for 10 minutes and a one-minute rinse with buffer. The polymer was added and allowed to sit for 15 minutes, followed by a two-minute rinse with buffer solution. DAB was added to the solution for 10 minutes, with a subsequent 2-minute wash using a buffer. Hematoxylin was utilized as a counterstain. The tissue was washed with buffer for two minutes, followed by a wash in water. It was then dehydrated, cleared, and mounted.
The immunostained sections were examined using a BZ-X710 microscope (Keyence), then photographed by an Axio CCamMR camera (Zeiss) [24].
Morphometric measures
H&E-stained and immune-stained slides from each group were used for measuring the following:
(1) The percentage % number of ovarian follicles in neonatal rats of 1st experiment (PND 4); by using H&E-stained sections, the ovarian follicles were classified, based on their stages, as the primordial (the oocyte is closely surrounded by a single layer of squamous granulosa cells) and early primary follicles (the growing oocyte is surrounded by a single layer of cuboidal cells or a multilayered mass of granulosa cells together with theca cells). The average number of primordial and primary follicles was assessed using the Olympus microscope CAST 2 stereological software v2.1.4. The stained slide glasses were scanned by the Nano Zoomer 2.0 RS virtual slide scanner (Hamamatsu Photonics). The follicles were counted at 7-section intervals (whole ovaries were sectioned, resulting in at least 50 sections per ovary). The number of numerous developmental follicles was multiplied by 7 to assess the total number of follicles per ovary [25]. Only follicles with visible oocytes were counted to prevent double counting.
(2) The average number of healthy ovarian follicles, atretic follicles, and corpus luteum in the pubertal rats in the 2nd experiment: the number of various developing follicles and atretic follicles was counted every five sections in high power fields (×100) multiplied by five to determine the total number of follicles per ovary. The follicles observed were classified into four groups: (a) primary follicles (as previously described in experiment-1), (b) secondary follicles (the oocyte is surrounded by several layers of granulosa cells together with theca cells, (c) growing follicles (pre-antral) as secondary plus a single large antrum, and (d) Graafian (antral) follicles (the oocyte is surrounded by cumulus oophorus cells, and is adjacent to a single large antrum. A follicle that either contains a fragmenting oocyte, surrounded by five or more layers of granulosa cells, or has a well-defined single follicular cavity containing cell debris was scored as nongrowing or atretic [26, 27].
(3) The average diameter of the counted ovarian follicles in both experiments and corpus luteum: five microscopic fields at a magnification of 10× were randomly chosen from each rat group. The ocular micrometer of a light microscope (Olympus EH; America Inc.) was used for measuring the diameter of each ovarian follicle, atretic follicles, and corpus luteum [28]. To avoid recounting the same follicle, only individual follicles, containing an oocyte with a nucleus, were evaluated [29]. The results are presented as the mean and standard deviation.
(4) The average area % of immune expression for Ki-67 positive and ARs in granulosa cells in both experiments. The immune-stained slides were photographed using an Olympus digital camera (E24-10 M pixel; Olympus) attached to an Olympus microscope with a ×0.5 photo adaptor. The obtained images were analyzed using Video Test Morphology computer software (VideoTesT) with a specific routine built-in for calculating an expression’s calibrated distance and area percentage. The results were shown as the ratio between the immunostaining positive area and the total area of the image [30].
Statistical analysis
Data from all groups were expressed as mean±SD. The initial tests were conducted on the normal data of control groups. The differences in the values between the study groups were measured using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Results were deemed statistically significant when the P-value was less than 0.05. The obtained data were analyzed using SPSS software, version 19 (IBM Co.).
Results
Biochemical results
Serum levels of the neonatal rats (PND 4) revealed a highly statistically significant reduction in the levels of the LH, FSH, estrogen, and progesterone (P<0.001) in the GA3-treated group in comparison with the control and Se-treated groups. There was a significant improvement in these hormonal levels when rats received Se alongside GA3 (P<0.05) compared to the control group (Fig. 1A). The same results were reported in the 2nd experiment of the pubertal rats regarding FSH and progesterone levels, meanwhile, the levels of LH and estrogen were significantly increased to mimic the control group in the Se-protected group (Fig. 1B). The data from both experiments of neonatal and pubertal rats are presented in Table 1.
Ovarian oxidative stress marker results
The ovarian level of the MDA (the marker of lipid peroxidation) of the GA3-treated group of both neonatal and pubertal rats showed a high statistically significant increase compared with control and Se-protected groups (P<0.001). But significant decrease in the activities of the antioxidant enzymes, SOD, and CAT was recorded in the same group compared to other study groups (P<0.001). On the other hand, the neonatal and pubertal rats that were co-treated with Se showed a significant decrease in MDA and an increase in antioxidant markers (SOD, CAT) (P<0.05) when compared to control groups. The data from both experiments of neonatal and pubertal rats are presented in Table 2.
Ovarian histology
H&E results of the neonatal rats
Ovarian sections of the control groups of the neonatal rats (PND 4) showed normal structural development. The ovary is covered externally by a layer of coelomic epithelium. Many primordial follicles were noticed in the ovarian cortex, each consisting of an oocyte surrounded by a single layer of squamous follicular cells. Early-developed primary follicles were observed in the cortex, where each oocyte was surrounded by a single layer of cuboidal cells (Fig. 2A). The GA3-treated rats showed disturbance and delayed developed ovaries. The ovary was covered by a thin and sometimes damaged coelomic epithelial layer. The ovarian cortices showed a small number of primordial follicles. A different shape of ovigerous cords with germ cell nest breakdown or collapsed germ cells was still present at this stage of development. Multiple spaces encircle the ovigerous cords (Fig. 2B, C). The H&E examination of the ovarian sections in the Se-protected group showed significant improvement in developmental events, with the ovaries covered by an intact coelomic epithelial layer. The cortex showed greater primordial follicles than the treated group. Early primary follicles with a small number of ovigerous cords with germ cell nest breakdown were observed in this group (Fig. 2D).
H&E results of the pubertal rats
Ovarian sections of the control groups of the pubertal rats showed normal ovarian structures with normal stages of follicular development in the cortex and with normal medulla. The follicles are; primary follicles, secondary follicles, growing follicles, and a well-developed Graafian follicle that showed a rounded oocyte surrounded by zona pellucida, zona granulosa cells, theca externa cell layer, and had follicular antrum. The Corpus luteum contained normal luteal cells (Fig. 3). The ovaries of the GA3-treated group showed a reduction in ovarian follicle numbers, abnormally shrinkage primary follicles, abnormally small secondary follicles with a greater number of degenerated (non-growing); atretic follicles having multiple layers of granulosa cells with degenerated or shrinkage oocytes with perifollicular cellular degeneration which were marked as spaces. Severe medullary congestion, hemorrhage, vacuolations, and congested blood vessels were observed. Other sections showed completely degenerated cortical follicles, necrosis with interstitial hemorrhage, edema, and excessive vacuolations. Abnormally elongated growing follicles with lens-like oocytes and a small antrum were observed (Fig. 4).
The ovarian sections of the Se-protected group showed nearly normal stages of follicular development beginning from the primary, secondary, growing follicle, Graffian follicle, some atretic follicles, and slightly degenerated secondary follicles. A small amount of medullary congestion, hemorrhages, and small areas of parenchymal tissue damage are still present. The corpus luteum showed normal and vacuolated luteal cells (Fig. 5).
Masson’s trichrome stain results
Examination of Masson’s trichrome stain of the ovarian sections of the control groups of the neonatal rats (PND 4) showed normal ovarian cellular stroma with scattered small thin collagen fibers only around blood vessels (Fig. 6A), but of the neonatal GA3-treated rats showed moderate fibrosis with deposition of thin collagen fibers that were more evident around ovigerous cords and in the coelomic epithelial layer (Fig. 6B). Meanwhile, the Se-protected group showed mild fibrosis with deposition of thin collagen fibers around primary follicles and within interstitial tissues (Fig. 6C).
Masson’s trichrome stain of the ovarian sections of the control groups of pubertal rats showed normal ovarian cellular stroma with scattered deposition of thin collagen fibers-stained green color around blood vessels with no fibrosis (Fig. 7A). Sections of the GA3-treated pubertal rats showed excessive fibrosis and deposition of thick collagen fibers that are more evident around blood vessels in interstitial tissues and the degenerated follicle (Fig. 7B). After Se coadministration, the ovarian sections of the pubertal rats showed mild fibrosis with deposition of thin collagen fibers around blood vessels and in interstitial tissues (Fig. 7C).
Immunohistochemical results
Ki-67 immuno-proliferative marker results
Ovarian sections of the studied groups stained with immuno-histochemical staining for Ki-67 showed strong and discrete staining of the cell nuclei of the granulosa cell of the primordial follicle and early primary follicles of control groups of the neonatal rats (PND 4) (Fig. 8A) but showed faint staining for Ki-67 in the germ cells in the ovigerous cords and mild stain in the follicular cells of the primordial follicle in GA3-treated rats of the same age group (Fig. 8B). However, after Se- co-administration, the ovarian sections showed moderate Ki-67 stain in the primordial follicle and strong stain in granulosa cells of early primary follicles which indicates the proliferative effect of the Se (Fig. 8C).
Immunohistochemical staining for Ki-67 of the ovarian sections of the pubertal control rats showed discrete positive brown staining of the cell nuclei of the follicular cells of the primary follicle and strong brown staining of the granulosa cell growing follicle (Fig. 9A), meanwhile, it showed faint brown staining of the follicular cells of the primary follicle and mild staining the granulosa cells of the atretic follicle in GA3-treated group (Fig. 9B). Sections of the Se-protected rats of the same age group showed mild staining in the follicular cells of the primary follicle but strong brown staining in the granulosa cells of the growing and Graafian follicles (Fig. 9C).
Androgen receptor expression results
The ovarian sections of the control groups of the neonatal rats (PND 4) stained with AR antibodies showed high expression in the granulosa cells of both primordial and early primary follicles (Fig. 10A). The GA3-treated group showed mild expression in the primordial follicles but a faint expression in the broken germ cell nests (Fig. 10B). Meanwhile, the Se-protected group showed moderate expression in granulosa cells of the primordial follicle but showed high expression in the early primary follicle (Fig. 10C).
Results of immunohistochemical staining of the AR of the control group of the pubertal rats showed high expression in the granulosa cells of primary follicles and slightly and sporadic expression of the theca cells of the growing follicles (Fig. 11A, B). Sections of the GA3-treated rats of the same age showed mild expression of AR in the follicular cells of the primary follicles and very faint expression of granulosa cells of the atretic follicles (Fig. 11C), After Se co-administration, the ovarian sections from the same age group showed high expression of ARs in the granulosa cells of the primary follicle and the theca cells of the growing follicle. (Fig. 11D).
Morphometric results
The measuring of the percentage % number of ovarian follicles of the neonatal rats (PND 4); showed a highly significant decrease in the average percentage of the primordial and early primary in the GA3-treated group in comparison with other study groups (P<0.001), then showed a significant increase again (P<0.05) after Se combined treatment in comparison with control group (Fig. 2E). The average diameter of primordial and early primary in the ovarian sections of the neonatal rats showed a highly significant decrease in the GA3-treated group in comparison with other groups (P<0.001) while showing a statistically significant increase in Se-protected rats of the same age (P<0.05) in comparison with the control group (Fig. 2F).
When counting the average number of ovarian follicles (primary, secondary, growing, Graafian, atretic, and corpus luteum in ovarian sections of the pubertal rats; a highly statistically significant low number of all follicles except for atretic follicles which showed a significantly higher number were found in the GA3 treated group (P<0.001) in comparison with other groups. The diameters of the ovarian follicles also were significantly decreased in this group (P<0.001) compared to different groups. The Se-protected group showed a statistically significant increase in ovarian follicles number except for the atretic follicles which showed a significant decrease compared to the control group (P<0.01). The diameters of the primary and the growing follicles were increased in the Se-protected group with insignificant differences with control. The secondary, the Graffian follicles and Corpus luteum diameters in Se-protected increased than GA3-treated but showed significant differences from the control group (P<0.05). Meanwhile, the diameters of the atretic follicles in this group showed a high statistical difference compared to the control (P<0.01). The values of the number and diameters of the ovarian follicles of the pubertal rats are presented in Tables 3 and 4 respectively.
Quantification of the average area % for counting the positive Ki-67 of granulosa cells in the ovarian sections of both age groups’ experiments (neonatal and pubertal rats); showed a statistically significant decrease in the GA3-treated group compared with the other study groups (P<0.001). Meanwhile, the Se-treated group showed a high significance increase compared with GA3 and control groups (P<0.001, P<0.05) respectively (Fig. 8D for neonatal and Fig. 9D for pubertal rats). The same results were reported regarding the average area % of immune expression for AR of the granulosa cells in both neonatal and pubertal rat groups (Fig. 10D for neonatal and Fig. 11E for pubertal rats).
Discussion
Ovary development begins early during fetal life in mammals and proceeds for a long time. Normal ovarian development involves a balance between the proliferation and apoptosis of germinative cells, which is the major phenomenon in establishing the maximal reproductive potential of adult females. Exposure to any toxicity during intrauterine life or folliculogenesis may influence the normal progression of development of the fetal ovary and the future of adult fertility [31]. GA3 is a substance whose molecular structure is similar to that of steroid hormones and may be an environmental endocrine disruptor [32]. Se supplementation has been proven to be protective against an extensive range of harmful factors, both chemical, such as drugs exerting severe side effects, heavy metals, carcinogens, mycotoxins, or pesticides, and physical, such as heat stress or magnetic fields [33]. This work represents an experimental demonstration of the potentially harmful effect of GA3 exposure on the pre and postnatal development of the rat ovary and to investigate the protective effect of Se.
Our biochemical findings of this study revealed a significant reduction in the serum levels of LH, FSH, estrogen, and progesterone in GA3-treated groups in both experimental ages which improved after Se administration, especially in the pubertal rats. Kamel et al. [34], reported in their studies that GA3 has a molecular structure similar to that of steroid hormones and possesses androgenic properties that make it one of the environmental endocrine-disrupting chemicals that can affect reproductive development by exerting estrogenic/anti-androgenic and androgenic/anti-estrogenic effects or by directly acting on the hypothalamic-pituitary-gonadal axis producing negative feedback mechanism on endogenous production of LH, FSH, estrogen, and progesterone levels. On the other hand, previous studies have also reported that GA3 exerts several estrogenic hormone-like effects accelerating the growth of animals and causing weight increases in the thyroid, ovaries, and adrenal glands suppressing them to produce androgens [35]. An explanation reported by Guo et al. [32], that GA3 exposure increased granulosa cell apoptosis, thus affecting follicle development and decreasing their hormonal production. The serum hormonal levels improved by Se administration due to its antioxidant and protective role against drug toxicity, decreasing the apoptotic process in the developing ovary [33]. Akintola et al. [36], demonstrate in their research that the synthesized Se nanoparticles restored estrogen and progesterone levels, as well as LH, FSH, testosterone, aromatase, and 17β-hydroxysteroid dehydrogenase levels which were altered in the hypertensive polycystic ovary syndrome rats.
In the current study, GA3 toxicity increased lipid peroxidation (MDA) in ovarian tissues and decreased the activities of the antioxidant enzymes (SOD and CAT). This may indicate that GA3-induced ovarian toxicity involves an oxidative effect and disturbs the balance between the antioxidant capability and harmful products in the ovarian tissues. This finding is consistent with the results of previous research [37, 38]. However, after Se co-treating, a significant decrease in MDA and an increase in antioxidant markers (SOD, CAT) were observed as Se increased the tissue antioxidants and decreased lipid peroxidation. These results align with Nady Ouais and Hassan [39] who assessed the Se antioxidant activities against carbon tetrachloride-induced liver damage.
Light microscopy results of GA3-treated neonatal rats revealed disruption of ovarian development, with fewer primordial follicles and ovigerous cords containing germ cell breakdown. These findings led to a significant decrease in the average percentage number of primordial and nearly absent early primary and their diameters as recorded in our morphometric measures of this study. Also, there were multiple histopathological changes in the same group of pubertal rats with abnormal development of the different stages of folliculogenesis, especially the growing follicles that led to a significant decrease in the average number and diameters of developing ovarian follicles except for atretic follicles which showed significant increase. These histological changes were attributed to the harmful effects of GA3 linked to oxidative stress, with ROS oxidizing and attacking vital cellular components, which can initiate or worsen cellular damage as reported in previous research [40]. This is in agreement with Bushra and Shenouda who documented the lung damage caused by other factors such as impairment of the antioxidant enzyme system due to lipid peroxidation caused by GA3 free radicals, which reduce the cellular defense system, and produce free radicals that account for the histo-pathological alteration of the different tissues [41] as occurs in the ovarian tissue of pubertal rats. Exposure to GA3 induces damage to the cellular DNA due to oxidative stress suppressing the intracellular growth factors responsible for follicular development [42]. GA3 toxicity interrupts the gonadal hormonal production considered an important factor in the development of the ovarian follicles; it also, increases the occurrence of apoptosis of granulosa cells, thus affecting the folliculogenesis. The same changes were noticed in rat testes of previous histological studies that showed loss of germ cells, derangement of the germinal cells, reduction in the size of the seminiferous tubules, and dystrophy of Leydig cells in rats treated orally and intradermally for 45 days with GA3 [43].
In this experiment, co-treatment with Se in both developmental stages reduced or eliminated the histopathological changes caused by GA3 toxicity as confirmed by the morphometric measures. In concurrence with these findings, Shen et al. [44] reported that Se administration positively modulates the growth and reproductive system development of the offspring in rats exposed to lead toxicity. Se is essential for female reproductive function, influencing follicle growth, maturation, and dominance in both cows and human [45, 46]. Grazul-Bilska and colleagues [47] have reported that low Se levels in a maternal diet suppress the growth of primordial, secondary, and antral follicles, stroma, and blood vasculature in the ovaries of the sheep fetus. Wang et al. [48] found that Se and vitamin E stimulate granulosa cell proliferation by reducing ROS levels, enhancing estradiol (E2) and progesterone (P4) secretion, regulating folliculogenesis-related genes, suppressing apoptosis, and increasing tissue antioxidant capacity, thereby improving redox balance in bovine granulosa cells.
In this experiment, ovarian Masson’s trichrome stain revealed deposition of thin collagen fibers around ovigerous cords and in the coelomic epithelial layer of neonatal rats treated by GA3 and excessive deposition of thick collagen fibers (a sign of fibrosis) around blood vessels in interstitial tissues. Collagen fiber deposition can be induced by ROS, which trigger the pro-fibrotic transforming growth factor-β, leading to increased fibroblast proliferation and ovarian fibrosis. Alsemeh et al. [4] explained the occurrence of fibrosis after sub-chronic GA3 treatment resulting from lipid peroxidation that led to protein and nucleic acid damage that eventually enhanced collagen formation in the livers of pregnant albino rats and their offspring.
The amount of collagen fiber deposition showed a significant decrease in this work after Se co-administration. Varlamova et al. [49] explained in their research that Se exhibits an anti-fibrotic effect by reducing nearly all pro-fibrotic and pro-inflammatory genes. Kreindl et al. [50] showed that Se compounds reduced fibrosis-related gene expression, including α-smooth muscle actin, collagen, TGF-β1, and TIMP-1, by inducing apoptosis in activated hepatic stellate cells. Se can lower the number of collagen-producing stellate cells and increase collagen degradation to inhibit fibrosis in fatty liver-associated disorder [51].
Anti Ki-67 antibody is a mouse monoclonal antibody that detects the Ki-67 protein considered a marker of cell proliferation. Ki-67 is positive during normal stages of folliculogenesis. It is observed in germ cells within nests and follicles, as well as in granulosa cells of developing follicles and stromal cells [52]. Our results of Ki-67 immunohistochemical examination of the GA3-treated rats showed faint staining for Ki-67 in the germ cells in the ovigerous cords and mild stain in the follicular cells of the primordial follicle. The same results were noticed in the pubertal rats which showed faint staining of the follicular cells of the primary follicle and mild staining of the granulosa cells of the atretic follicle. In agreement with our findings, Khadrawy et al. [38] found that GA3 exposure impairs ROS scavenging, causing damage to DNA leading to enzyme inactivation, genotoxicity, cytotoxicity, and membrane integrity loss that explain the toxic effects of GA3 on the proliferating capacity of ovarian follicles. The same explanation reported by Ebrahim et al. [53] in their study that oxidative stress can cause inhibition of cellular proteins responsible for cellular proliferation as Ki-67 which supports the hypothesis that oxidative stress is linked to the attenuation of cellular proliferation and the morphological dysfunction observed in the ovarian tissue that is induced by the SOF treatment. The results of the Ki-67 expression study improved after the coadministration of Se in both experimental ages, indicating Se’s effect on cell survival. Our results were consistent with previous studies that reported the potential antioxidant role of seleno-proteins on follicle dominance protecting the dominant follicle from increasing levels of ROS suggesting promotion of follicular cell proliferation [54]. Our results of Ki-67 immune expression were confirmed by morphometric measures of quantification of the average area % for counting of positive Ki-67 of granulosa cells in both experimental ages; that showed a statistically significant decrease in GA3 treated groups but showed a high significant increase in the groups that were co-administrated with Se.
ARs are found in all three components of the ovarian follicle; oocytes, theca cells, and their protein are most in granulosa cells which contain abundant amounts of AR RNA and its protein [55]. Laird et al. [56] showed that the androgens stimulate the growth of both preantral and antral follicles in various species. In this study, GA3 toxicity affects ARs expression in both developmental ages. According to previous studies; ROS of GA3 attack biomolecules such as DNA, lipids, proteins, and protein transcriptional factors causing enzyme inactivation, and RNA protein suppression, thus affecting protein stability and gene expression [57].
The results of ARs expression were increased after Se co-administration as confirmed by measuring the average area % of immune expression for ARs. Basini and Tamanini [58] demonstrated that Se might regulate the growth of the granulosa cells and 17β-estradiol bio-synthesis in adult ovaries in vitro, promoting AR expression. Previous studies have also elucidated that Se and selenoprotein levels are important for healthy follicles and perform a vital antioxidant function during early and later growth and the proliferation of the follicles [54].
In conclusion, the results of the current study showed that prenatal (maternal) exposure of female rats to GA3 or postnatal (pre-pubertal exposure) exhibited delayed & breakdown of the developmental process of folliculogenesis in the rat’s ovaries, as evidenced by multiple histological alterations, immunohistochemical levels, and morphometric measures of the developing ovarian follicles. Co-administration of Se minimized the developmental alternation, as confirmed by histological, immunohistochemical, and morphometric measures. The study recommended considering Se as a protective agent against the hazardous effects of plant regulators that are widely used and reach the developing organs.
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