Evaluating the effects of quinestrol on the reproductive organs of the Nile rat (Arvicanthis niloticus) for use in the rat control

ABSTRACT The purpose of this research was to determine whether the synthetic estrogen quinestrol has antifertility effects on male and female Nile rats (Arvicanthis niloticus). Both male and female rats were orally administered quinestrol dissolved in castor oil at a dosage of 1 mg/kg for seven days. In contrast, male and female control rats were given castor oil alone. The use of this dosage of quinestrol resulted in a decrease in the weight of reproductive organs in both sexes, as well as a decrease in sperm count and motility and an increase in the percentage of abnormal sperm. Various histopathological alterations were observed in the testicular, epididymal, and ovarian tissues. Significant reductions in immunohistochemical markers such as androgen receptor protein (AR) and Wilm’s tumor nuclear protein 1 (Wt-1) were observed in treated male rats. These results indicate that quinestrol induces infertility in both males and females of the Nile rats. Therefore, it is recommended for use in integrated pest management campaigns targeting these serious vertebrate pests.


Introduction
Traditional lethal methods of controlling vertebrate pests, such as rats and mice, have proven to be inefficient in eradicating them.Rodents, which are short-lived and fast-breeding pests, can quickly restore their numbers through reproduction [1].Additionally, the poisons used in these methods contribute to environmental pollution and face increasing opposition from animal welfare groups [2].These issues have led vertebrate pest managers to seek alternative approaches for controlling these serious pests.One such approach is the use of anti-fertility compounds, which are more humane and environmentally safe [3,4].
The Nile rat, Arvicanthis niloticus, poses a significant threat to crops such as wheat, rice, maize, sugar cane, beans, vegetables, and fruits However, it is most prevalent in grain fields [5,6].It is believed that this pest harbors microorganisms that can cause illnesses in both humans and plants, such as bubonic plague and rice yellow mottle virus [7].The distribution of this vertebrate pest covers a wide geographical area, including several African countries and southwestern Arabia.In Egypt, areas such as the Nile Valley and Delta, El-Faiyum, Dakhla, Khargra Oases, and El Maghreb, as well as canals stretching into the western Mediterranean coastal desert, are affected by this distribution [8,9].
Quinestrol is an artificial estrogen that has antifertility effects similar to a wide variety of human oral contraceptives.Various rodent species, including Brandt's voles (Lasiopodomys brandtii), Sprague-Dawley rats, Himalayan field rats (Rattus nitidus), house mice (Mus musculus), and Natal multimammate mice (Mastomys natalensis), have been found to be infertile after exposure to different doses of this compound [10,11,12,13,14].It is well known that the doses of quinestrol required for antifertility vary among different mammalian animals [15].Interestingly, the use of estrogen has a greater antifertility impact on males compared to androgens [16].In addition to its high efficacy as a rodent contraceptive, its antifertility effects last for a long time.Quinestrol causes damage to the main reproductive organs in both sexes.The ovary plays two main roles in females: ova production for fertilization by male sperm and functioning as an endocrine gland that aids in fertility [17].The epididymis, on the other hand, is the final stage of the sperm maturation process, during which spermatozoa become motile and gain the ability to fertilize oocytes.Furthermore, the epididymis stores mature sperm [18].
The spermatogenic niche is crucial for spermatogonia to produce viable sperm.Sertoli cells, which directly interact with germ cells, are unique among these cells and play a crucial role in spermatogenesis.Malfunctioning of these cells often leads to spermatogenic failure [19].In the seminiferous tubules, Sertoli cells provide nourishment and support for germ cells, as well as contribute to spermatogenesis and testis development.Sertoli cells can only support a limited number of germ cells, and the daily production of sperm cells is positively correlated with the presence of these cells [20].Different cells express distinct markers, some of which are unique to their specific cellular group.Wilms' tumor (WT-1) and androgen receptors (AR) are the markers frequently expressed by Sertoli cells.
To the best of our knowledge, quinestrol has not been previously used as an antifertility agent in Nile rats.Therefore, an experimental dose of this compound was tested for its antifertility effects on Nile rats.The current study examined the antifertility effects of quinestrol on Nile grass rats, Arvicanthis niloticus, of both sexes.Various morphometric, histological, and immunohistochemical methods were used to compare the testes, epididymis, and ovaries of treated and untreated rats.

Chemicals
From Sigma-Aldrich Chemical Co.(St.Louis, MO, USA), the quinestrol used in the current study was purchased.Before administering the chemical to test animals, it was dissolved in the appropriate amount of castor oil.Unless otherwise noted, all chemicals and reagents used here were of high analytical quality and obtained from El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt).

Experimental animals
In total, 60 mature Nile rats (Arvicanthis niloticus; 30 males and 30 females), each weighing between 120 and 200 g, were employed in the experiment.The Nile rats were gathered from farms in Giza Governorate, Egypt, and then transported for nearly an hour to the animal house at the Zoology Department, Ain Shams University in Abbasiya, where they were kept in metal cages (20 cm x 20 cm x 30 cm).They were acclimated to laboratory settings for 10 days before being housed in a 22-23°C, 50-55% humidity environment with a 12-hour light/dark cycle.The treatment of the Nile rats was humane, and they were allowed unrestricted access to food (commercial food pellets) and water.The Ain Shams University Research Ethics Committee approved this study protocol with an approval code (ASU-SCI/Zool/2022/12/2).

Rat experimental groups
Using an electronic balance, the starting body weight of 60 adult Nile rats was determined, and they were randomly divided into four groups, with 15 rats in each group.They were further divided into two male groups and two female groups, as follows: Group I (male control group): Each male was orally given only 1 mL of castor oil per kg (body weight) per day for 7 consecutive days.
Group II (male experimental group): Each male was orally given 1 mg of quinestrol (dissolved in castor oil)/kg (body weight)/day for 7 consecutive days (0.18 mg/g dissolved in 1 mL of caster oil for males) [13].
Group III (female control group): Each female was orally given only 1 ml of castor oil per kg (body weight) per day for 7 consecutive days.
Group IV (female experimental group): Each female was orally given 1 mg of quinestrol (dissolved in castor oil)/kg (body weight)/day for 7 consecutive days (0.125 mg/g dissolved in 0.6 mL of caster oil) [13].

Necropsy and sample collection
All rats were euthanized using chloroform 24 hours after the last dose, and their body weight was measured (final body weight).The weight gain or loss percentage was determined by the following formula: {(final weight-starting weight)/ (starting weight)x 100} [2].The left cauda epididymis of male rats was excised, removed, and stored in 2 ml of phosphatebuffered saline at 37 degrees Celsius for later sperm evaluation [21].The remaining epididymis was quickly fixed for histological analysis.Male and female reproductive organs were removed, cleaned of excess fats and connective tissues, and weighed in grams.This included the testes, epididymides, seminal vesicles, and prostate glands in males and the uterus and ovaries in females.The formula (organ weight/ body weight) x 100 was used to calculate the reproductive organ index (relative weight) [2].The percentage of weight change in organs was determined by multiplying the result of the following calculation by 100: treatment organ weight -control organ weight)/control organ weight [12].The right testis, epididymis, and ovary of each experimental rat were fixed in 10% neutral buffered formalin before being processed for histological analysis.

Sperm characteristics
To allow the spermatozoa to release, the cauda epididymis of each rat was dissected, minced into 2 ml of 0.9% phosphate-buffered saline, and incubated at 37°C for 30 minutes.The total number of sperm was measured using a Neubauer-hemocytometer and reported as sperm per mL X10 6 .The percentages of motile sperm were calculated using a light microscope with a magnification level of 400X, following the procedures of Taha and Soliman [4].Sperm morphology was estimated by smearing sperm on microscope slides, allowing them to air dry for about a minute, fixing them in methanol, and staining them with eosin [3].One hundred spermatozoa from each sample were examined by a light microscope with an oil immersion lens at 1,000X [4].The sperm morphology was assessed according to WHO standard protocols [22] and identified as abnormal or normal.Sperm abnormalities were expressed as a percentage of the total number of sperm observed [4].

Histological procedures
Testicular, epididymal, and ovarian samples were collected, fixed in 10% neutral buffered formalin for at least 10 hours, rinsed in running tap water for 12 hours, and dehydrated in ascending concentrations of ethyl alcohol.Paraffin wax blocks were made from tissue samples previously cleaned with xylene.Next, we used a Yidi semi-automated microtome to cut the tissues into 5 µm-thick sections.Using hematoxylin and eosin staining, tissue slices were stained and then mounted on slides [3].Slides were viewed and photographed using a Leica microscope and Leica camera (LEICA, ICC50 HD) at the Cairo University Research Park, Faculty of Agriculture, Cairo, Egypt.

Morphometric measurements
Male rats were examined to determine the following: Firstly, in testicular tissue, 1) the mean thickness of the tunica albuginea, and 2) the mean diameter of seminiferous tubules 3) the mean height of the germinal epithelium Second, in the epididymal tissue, 1) the mean diameter of the epididymal ducts; 2) the mean diameter of the lumen of the epididymal ducts; and 3) the mean thickness of the epididymal ducts.All these measurements were analyzed semi-quantitatively using an ocular micrometer calibrated with a stage micrometer [23].At least 10 randomly chosen tubular profiles that were round or nearly round were measured for each rat at a magnification of 400 × .The total number of ovarian follicles was counted in females.

Johnsen's score of spermatogenesis
Johnsen's score was used to determine the extent of injury to the testis [24].A scoring system with ten points was developed to assess spermatogenesis along the profile of cells in the seminiferous tubules.Each tubule region receives a score between ten and one on this scale.A Johnsen score of ten represents maximum spermatogenesis activity, while a score of one indicates a lack of any germ cells.The ten criteria used in this are shown in Table 1.The mean final value (at least 10 seminiferous tubules per rat) indicates the final Johnsen score for each male rat.

Immunohistochemistry
To prepare for androgen receptor (AR) labeling or Wilms' tumor nuclear protein (Wt-1) staining, testicular and epididymal sections were rehydrated in 10 mM citrate buffer [pH 6 for 15 min] for AR or 0.001 mM EDTA buffer [95°C for 15 min] for Wt-1.The slices were incubated with primary antibodies at 4°C overnight after being immersed in 1% hydrogen peroxide for 10 minutes and 1% bovine serum albumin (BSA) for 30 minutes.Rabbit polyclonal anti-rat AR (SC-816; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit polyclonal anti-rat Wt-1 (SC-192; Santa Cruz Biotechnology, Santa Cruz, CA, USA) In the absence of primary polyclonal antibodies, nonimmune rabbit IgG was used at the same dose.Phosphate-buffered saline (PBS) was used to wash the sections, and then they were treated with goat anti-rabbit IgG coupled to horseradish peroxidase for 30 minutes at room temperature.Diaminobenzidine (DAB) was used as a substrate for the final visualization.Haematoxylin counterstaining and resin mounting followed ethanol and xylene dehydration of the slides [25,26].

Statistical analysis
Statistics were summarized by computing the mean and standard error of the mean (SEM).The t-test for independent samples was employed in GraphPad Prism TM (version 8.0) Table 1.Criteria used in the evaluation of spermatogenesis.

Scores
Evaluation system of spermatogenesis 10 Complete spermatogenesis 9 Disorganized epithelium, many late spermatids, and slightly impaired spermatogenesis 8 Few late spermatids and less than five spermatozoa per tubule 7 There are several early spermatids but no spermatozoa or late spermatids 6 There are no spermatozoa, few early spermatids, and no late spermatids 5 Lots of spermatocytes but no spermatozoa or spermatids 4 There are few spermatocytes and no spermatozoa or spermatids 3 Just spermatogonia 2 Sertoli cells exclusively; no germinal cells 1 Absence of seminiferous epithelium to compare the means, which followed a normal distribution (GraphPad, San Diego, CA).A significant level of P < 0.05 was used to determine statistical significance [25,27].

Effect of quinestrol on general body health and body weights of both sexes
Throughout the study period, no mortalities were recorded among the normal or treated rats.Treated males and females appeared active with normal behavior and appetite and without noticeable variations in body mass, compared to normal ones (Table 2).

Sperm characteristics
Sperm count as well as the percentage of motile sperm in quinestrol-treated male rats were significantly lower than the corresponding ones in untreated males.Compared to untreated males, treated ones showed a highly significant increase in the percentage of immotile and abnormal sperm (Table 3).

Gross morphology and relative organ weights
Rats given the treatment showed smaller and paler testicles, epididymides, and seminal vesicles than their control group.The relative weights of the testis, epididymis, and seminal vesicle were all significantly lower in the quinestrol-treated group compared to the control group (Figure 1a,b).However, there was no statistically significant difference between the treated and control groups in terms of the relative weight of the prostate gland (Table 4).

Histological, morphometric changes of testis and Johnsen's score
The testes of the control group displayed the typical histological structure of Nile rat testes on histological analysis (Figure 2a-c).Upon examination of the testicles of male rats treated with quinestrol, degenerative alterations in the seminiferous tubule tissues and a thicker fibrous tunica albuginea were observed.This thickening was confirmed through morphometric analysis (Figure 2d) (Table 5).Notably, there was marked tubular vacuolar degeneration, and spermatogonia lining the seminiferous tubules.Additionally, the intertubular spaces appeared to widen (Figure 2e).The testicular tissues exhibited vascular congestion, necrotic spermatogenic cells with low spermatogenesis activity (Johnsen's score), and lumina without spermatozoa (Figure 2e,f) (Table 5).The mean diameter of the seminiferous tubule and the height of their epithelial lining was significantly reduced (Table 5).

Histological and morphometric changes of epididymis
Epididymal ducts lined with pseudostratified columnar epithelium resting on basal lamina were seen in abundance under the microscope in the epididymis of untreated rats, along with abundant apparently healthy spermatozoa in their lumina (Figure 3a,b).However, when the epididymis of males treated with quinestrol was examined, very few spermatozoa along with much cellular debris were detected (Figure 3c,  d).The primary cells lining the epididymal ducts displayed vacuoles compressing the nuclei as well as some pyknotic nuclei (Figure3c,d).It was found that the blood vessels were congested (Figure 3c,d).The mean of epididymal ducts and  their lumens and the mean height of their lining epithelium were significantly reduced (Table 6).

Immuno-histochemical changes in testes
For androgen receptor (AR), the nuclei of Sertoli cells in the basal layer of the seminiferous tubules, the nuclei of several germ cells at different stages of development, and the nucleus and cytoplasm of elongating spermatids were all locations of AR in untreated rats.Sertoli and Leydig cell nuclei were the primary sites of AR positivity (Figure 4a,b).AR immunostaining was significantly reduced in these cell types and sites in males treated with quinestrol (Figure 4c,d).In contrast to untreated males, the mean count of AR-positive cells was significantly lower in the treated group (Figure 6a).Positively stained Sertoli cells with Wt-1 were equally distributed on the basal layer of seminiferous tubules, and the number of positive cells was almost present at various stages inside the seminiferous tubules in untreated males, indicating that Wt-1 is uniquely expressed in Sertoli cells (Figure 5a,b).For these cell types and sites,  the average number of Wt-1 positive cells was significantly reduced in males given quinestrol (Figure 5c,d).The mean count of Wt-1 positive cells showed a significant decrease in the quinestrol-treated males, compared to the untreated ones (Figure 6b).

Immuno-histochemical changes in epididymis
In normal males, AR was localized in the nuclei of both tubular epithelial cells and intertubular spaces (Figure 7a,b).In quinestrol-treated males, the count of AR-positive  cells decreased markedly in these cell types and locations (Figure 7c,d).The mean count of positive cells showed a high significant decrease (P < 0.001), compared to the control group (Figure 9a).Wt-1 is strongly expressed in normal males through the epithelial lining of epididymal ducts (Figure 8a,b).In quinestrol-treated males, the Wt-1 positive cells decreased markedly in these locations (Figure 8c,d).The mean count of Wt-1 positive cells in the epididymis of treated males showed a significant decrease (P < 0.001) in these cell locations, compared to the control group (Figure 9b).

Gross morphology and relative organ weights
The ovaries of quinestrol-treated females were paler in color and smaller in size than those of untreated females.A significant decrease in the relative weight of the ovaries of treated females, compared to untreated ones, P < 0.001 was observed (Table 7).The uteri of the quinestrol-treated females showed marked edema and erythema (Figure 10b,c).There was a non-significant change in the relative     weight of the uterus of the quinestrol-treated group compared to untreated ones (Table 7).

Histological and morphometric changes of ovaries
The ovaries of untreated females showed normal organization of the cortex and medulla with wide vascularization.Various types of follicles, such as primordial follicles, antral follicles, atretic follicles, and Graffian follicles could be observed in the cortex (Figure 11a).In quinestrol-treated females, developing follicles were significantly destroyed, resulting in a decrease in the number of follicles.
Additionally, the number of corpora lutea and congested ovaries increased (Figure 11d), and the size of oocytes shrunk (Figure 11e), compared to the control group (Figure 11b).The total number of follicles in the treated females decreased significantly (Figure 12) compared to the untreated group (P < 0.01).

Discussion
The Nile rat, Arvicanthus niloticus, is a significant vertebrate pest that experiences periodic outbreaks due to its high population density reaching 1 individual/100 m 2 or more [5].Greaves [5] also noted that this rat has caused average crop losses in Egypt and the Sudan amounting to 20-25% or more.Anticoagulant rodenticides, and a lesser extent, zinc phosphide, were previously used to control this rat [6].
In this current study, the contraceptive effects of quinestrol were evaluated in both sexes of this rat from Egypt.This evaluation is based on three reasons:  (1) Quinestrol is a biodegradable compound [13] making it safe for non-target animals.(2) Its half-life ranges from as low as 18 hours [13] or approximately 1 day [28] to 16 days [29], depending on environmental conditions.
(3) It is known to be the most effective contraceptive compound at low doses [11].
The present study demonstrated contraceptive effects in both sexes of Nile rats at a low dose of 1 mg/kg for 7 consecutive days.This is in comparison to alpha-chlorohydrin which is effective at a dose of 85.7 mg/kg for 4 days in other vertebrate pests [4].
It is well known that changes in body weight are good indicators of the effect of tested chemical compounds on the general health of treated animals [2].After treatment, there were no significant changes in the body weights of treated males and females.They consumed normal daily amounts of water and food and exhibited normal behavior during the whole experimental period.Similar results in body weight were also observed in both sexes of the Himalayan field rat, Rattus nitidus when treated with the same compound [12].Therefore, quinestrol fulfills one of the important characteristics of an ideal contraceptive compound [3,30].
Changes in the weights of male reproductive organs are good measures for estimating the antifertility effects of tested compounds [31].When comparing the relative weights of the testis, epididymis, and seminal vesicle between treated and untreated males, the current research revealed that quinestrol significantly reduced the aforementioned relative weights in treated males.Other animal species treated with quinestrol showed similar effects in some reproductive organs [10][11][12][13].
Sperm count and motility are routinely used as indicators of the reproductive potential of males [3,4].The number of sperm in quinestroltreated male Nile rats was less than half (44.9%) the number of sperm in untreated rats.Sperm motility is also a useful measure in evaluating sperm fertilizing potential in males [32].In the present study, the percentage of motile sperm showed a significant decrease in treated males compared to untreated males.This result could be suggested to a decrease in sperm ATP content, which in turn causes an inhibition in sperm motility.Since ATP is important for the normal forward progressive movement of sperm, any slight decrease in its level would negatively affect sperm motility [33].Many experimental studies have shown that quinestrol treatment impairs male reproductive potential by reducing semen quality, sperm count, and motility [34,35].The morphology of sperms is also a good indicator of maturation and fertility in males [4,36].In the current study, sperm abnormalities significantly increased in quinestrol-treated males compared to untreated ones.Similar results were recorded in quinestroltreated Natal multimammate mice, Mastomys natalensis [14].The effect of quinestrol treatment on male reproductive organs and sperm characteristics could be attributed to the increased level of estrogen in treated males which could have a negative feedback effect on the hypothalamus-hypophyseal axis.This could ultimately result in decreased testosterone levels and impaired spermatogenesis in treated males [37][38][39].The maturation of sperm in the epididymis and the rate of seminal vesicle secretion were both affected by quinestrol treatment [34,35].
The testes of quinestrol-treated males exhibited a significant decrease in Johnsen's scores compared to untreated males, indicating the presence of testicular damage.The increased thickness of the tunica albuginea in treated males is explained by a decrease in testicular parenchyma volume [3,31,40].
Tubular shrinkage may, in turn, be attributed to the loss of germ cells [3,4] due to the disturbance injunctions between Sertoli and germ cells [31,41].There was also a significant reduction in the epithelial height of seminiferous tubules, compared to untreated males.Similar findings have confirmed the antifertility effect of quinestrol on males of the house mouse, Mus musculus [13].Quinestrol treatment also resulted in several histopathological and morphometric changes in the tissues of the epididymis, such as the formation of intraepithelial spaces and vacuoles, the presence of cellular debris inside the epididymal lumen, and a significant decrease in the thickness and diameter of epididymal ducts.Previous studies recorded several similar changes in the epididymis when using other chemicals [42,43].
Wilms' tumor (WT-1) protein is highly important for normal testis architecture and reproductive ridge survival indicating a potential developmental function in the genital system [25].The androgen receptor (AR) mediates the effects of androgens such as testosterone and dihydrotestosterone.The AR protein plays significant roles in the growth and maintenance of various systems, including the cardiovascular, immunological, neurological, musculoskeletal, and hemopoietic systems [26].
These histological findings were supported by the mean reactive cell counts of AR and Wt-1 immunoexpression.The presence of small numbers of positive cells is explained by the testicular and epididymal damage that occurred in these rats.
In the testis, AR-positive stained cells of round and elongated spermatids, nuclei of Sertoli and Leydig cells, and some germ cells were less than untreated males.After treatment immunohistochemical examination, revealed a reduction in the total number of positively stained Wt-1 cells in the tubules of treated males compared to control males.A previous study by Liu et al. [13] showed that quinestroltreated Musmusculus rats exhibited a slightly lower number of AR and Wt-1 positively stained cells compared to control males.
In epididymal ducts, a similar significant decrease in AR and Wt-1 positively stained cells was observed in testis tubules.Elbakry & Ibrahim [44] found that treatment of albino rats with the bactericide Triclosan (TCS) at a dosage of 200 mg/kg daily for 8 weeks resulted in a reduction in nuclear immunoreaction to AR in the lining of the epididymal ducts.
In females, quinestrol treatment had a negative effect on the relative ovary weight.Liu et al. [12] reported a 49% decrease in ovary weight, compared to untreated females in female Rattus nitidus treated with quinestrol.It seems that quinestrol treatment does not affect uterus weight, as observed in the present study and previous studies [10,12].However, an edematous swelling was seen upon inspection of the general morphology of the uteri.Possible explanations include the observed effects of elevated levels of estrogen and progesterone on uterine structure [45].Additionally, 8 and 40 days of quinestrol treatment resulted in uterine edema in female Mastomys natalensis [14].
According to the histological analysis,the number of follicles was signifcantly reduced, in the ovaries of quinestrol-treated females.on the other hand, there was a significant increase in the congestion of blood vessels.Ovarian dysfunction is associated with a decline in follicle count [46].

Conclusion
Based on the findings of the current investigation, quinestrol administration of both sexes of Nile grass rats for 7 days at a dose of 1 mg/kg had significant antifertility effects on them.Therefore, this compound is recommended for use in integrated pest management campaigns for this vertebrate pest in Egypt.

Figure 1 .
Figure 1.Photographs showing the morphological structure of the testis of male Nile grass rats.(a): normal testis with prominent blood vessels over their surface (blue arrows) and large vesicular seminalis (green arrows) (b) the testis of quinestrol-treated males had marked edema (blue arrows) and appeared smaller in size and length.The vesicular seminals were also too small to appear.

Figure 2 .
Figure 2. Photomicrographs of testicular sections from normal and treated Nile rat males stained with H&E.Normal males: (a) display a typical testicular structure with seminiferous tubules (ST) and a thin tunica albuginea (arrow); (b) shows regular seminiferous tubules (ST) with narrow intertubular spaces (IT); (c) is a magnified photo of seminiferous tubule with large spermatogenic cells (arrow), lumen filled with abundant spermatozoa (*), and narrow intertubular spaces (IT).Quinestrol-treated males: (d) exhibits deformed seminiferous tubules (ST) and a thickened fibrous tunica albuginea (arrow); (e) shows seminiferous tubules (ST) with marked tubular vacuolar degeneration, necrosis of the germinal epithelium lining the tubules and widening of the intertubular spaces (IT); (f) illustrates marked vascular congestion (vc), seminiferous tubules (ST) with many darkly stained shrunken germinal cells (arrows), and a lumen devoid of spermatozoa (*).

Figure 3 .
Figure 3. Photomicrographs of epididymal sections from normal males and quinestrol-treated males of Nile rats stained with H&E.Normal males: (a) and (b) show epididymal tubules (ET) lined by pseudostratified columnar epithelium (PE) and filled with spermatozoa (SM) in their lumina.There are narrow interstitial spaces between the ducts (IT).Quinestrol-treated males: (c) and (d) show shrunken epididymal ducts (ET) lined by pseudostratified columnar epithelium (PE).There are wide interstitial spaces between the ducts (IT), that are filled with cellular debris (star), a few spermatozoa are present (red arrow), along with vacuolated epithelial cells (thick arrow), some pyknotic nuclei (thin arrow), and vascular congestion (V).

Figure 4 .
Figure 4. Photomicrographs of testicular sections from male Nile rats, displaying representative immunohistochemical assays for the AR antibody and the immunolocalization of the nuclear androgen receptor (AR) antibody.Normal males: (a) and (b) exhibit strong immunohistochemical staining for AR.In (b), a magnified photo reveals that AR is primarily localized at the basement membrane (thin arrow), germinal cells (thick arrow), and intertubular space (IT).Quinestrol-treated males: (c) and (d) exhibit a weak AR immunoreaction.In (d), a magnified photo shows a higher number of necrotic degenerated seminiferous tubules with weak reaction in destructed germinal cells (thick arrow), basement membrane (thin arrow), and no nearby reaction in intertubular spaces (IT).

Figure 5 .
Figure 5. Photomicrographs of testicular sections from male Nile rats illustrating representative immunohistochemical assays for the Wt-1 antibody and immunolocalization of nuclear Wt-1 antibody.In normal males: (a) and (b) exhibit strong brown immunohistochemical stains for Wt-1.A magnified photo in (b) reveals that Wt-1 is primarily localized at the basement membrane (thin arrow), germinal cells (thick arrow), and intertubular space (IT).In quinestrol-treated males, (c) and (d) display a weaker Wt-1 immunoreaction.A magnified view in (d) shows necrotic degenerated seminiferous tubules, with a weak reaction in the destroyed germinal cells (thick arrow) and basement membrane (thin arrow), and nearby no reaction in intertubular spaces (IT).

Figure 6 .
Figure 6.Changes in the mean count of positive cells of immunostaining in the testis of Nile rats in the studied groups.a) Mean count of AR-positive cells.b) Mean count of WT-1 positive cells.Means and SEM are used to express values.P*-value indicated that the values were significantly different from those of the control group, where P*<0.005, and P**<0.001.

Figure 7 .
Figure 7. Photomicrographs of epididymal sections of normal and quinestrol-treated males of Nile rats showing immunohistochemical assays with immunolocalization of nuclear androgen receptor (AR) antibody.Normal males: (a) and (b) show an exclusively strong nuclear AR immunoreaction in the epididymal ducts.(b) a magnified photo showed that AR mainly localized at the basement membrane (thin arrow), germinal cells (thick arrow), and intertubular space (IT).Quinestrol-treated males: (c) and (d) show a weak AR immunoreaction (arrows).(d) showed a magnified photo of the number of tubules with weak reactions in destructed germinal cells (thick arrow), basement membrane (thin arrow), and nearby no reaction in intertubular spaces (IT).

Figure 8 .
Figure 8. Photomicrographs of epididymal sections of normal and quinestrol-treated males of Nile rats showing immunohistochemical assays with immunolocalization of nuclear (Wt-1) antibody.Normal males: (a) and (b) show an exclusively strong nuclear Wt-1 immunoreaction in the epididymal ducts.(b) a magnified photo showed that Wt-1 mainly localized at the basement membrane (thin arrow), germinal cells (thick arrow), and intertubular space (IT).Quinestrol-treated males: (c) and (d) show a weak Wt-1 immunoreaction (arrows).(d) showed a magnified photo of the number of tubules with weak reaction in destructed germinal cells (thick arrow), basement membrane (thin arrow), and nearby no reaction in intertubular spaces (IT).

Figure 9 .
Figure 9. Changes in the mean count of positive cells of immunostaining in the epididymis of Nile rats in the studied groups.a) Mean count of AR-positive cells.b) Mean count of WT-1 positive cells.Means and SEM are used to express values.P* value indicated that the values were significantly different from those of the control group, where P*<0.001.

Figure 10 .
Figure 10.Photographs showing the morphological structure of uteri of female Nile grass rats.(a): normal uterus with high blood supply (arrows), (b) uterus of quinestrol-treated females with marked edema (arrows), (c) uterus of quinestrol-treated females with remarkable erythema (arrows).

Figure 12 .
Figure 12.Changes in the mean total number of follicles in the ovaries of Nile rats in the studied groups.Means and SEM are used to express values.P* value indicates that the values were significantly different from those of the control group, where P*< 0.01.

Table 2 .
Effect of quinestrol on body weights of male and female Nile rats.

Table 3 .
Effect of quinestrol on sperm characteristics in the Nile grass rats.
Means and SEM are used to express values.P* and P** values indicated that the values were significantly different from those of the control group, where P*<0.01 and P**<0.001.

Table 4 .
Effect of quinestrol on male reproductive organs of male Nile grass rats.
The relative weight of reproductive organs (%)Means and SEM are used to express values.The P* value indicated that the values were significantly different from those of the control group, where P*<0.001.

Table 5 .
Effect of quinestrol on testicular morphometrics analysis and the stage of spermatogenesis in the Nile grass rats.

Table 6 .
Effect of quinestrol treatment on epididymal ducts and epithelium in the Nilegrass rats.

Table 7 .
Effect of quinestrol on reproductive organs of female Nile grass rats.