The Effect of Rutin on Progesterone and Estrogen Receptor Expression in Uterine Endometrial Tissue in the Heterotopic Transplantation of Newborn Mouse Ovary

AUTHORS

Tayebeh Hadigol 1 , Aligholi Sobhani 2 , Masoud Hemadi 1 , Saeid Nekoonam 2 , Alireza Shams 3 , Bahram Eslami Farsani 1 , Maryam Dastoorpoor 4 , Ghasem Saki ORCID 1 , *

1 Department of Anatomy, Faculty of Medicine, Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Anatomy, Faculty of Medicine, Alborz University of Medical Sciences, Karaj, Iran

4 Department of Epidemiology and Biostatistics, Menopause Andropause Research Center, Jundishapur University of Medical Sciences, Ahvaz, Iran

How to Cite: Hadigol T, Sobhani A, Hemadi M, Nekoonam S, Shams A, et al. The Effect of Rutin on Progesterone and Estrogen Receptor Expression in Uterine Endometrial Tissue in the Heterotopic Transplantation of Newborn Mouse Ovary, Iran Red Crescent Med J. Online ahead of Print ; 21(4):e86289. doi: 10.5812/ircmj.86289.

ARTICLE INFORMATION

Iranian Red Crescent Medical Journal: 21 (4); e86289
Published Online: May 6, 2019
Article Type: Research Article
Received: November 11, 2018
Revised: April 18, 2019
Accepted: April 21, 2019
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Abstract

Background: Rutin (quercetin-3-rhamnosyl-glucoside), a flavonoid, is derived from plants and has antioxidant properties.

Objectives: This study aimed to evaluate the effect of different concentrations of rutin on mouse ovary heterotopic allotransplantation.

Methods: The present animal experimental study was conducted on 40 female adult Balb/c mice weighing 30 ± 5 g at the Jundishapur University of Medical Sciences, Ahvaz, Iran, during 2016 - 2018. The mice were divided by permuted block randomization into 8 groups (n = 5): OVX (ovariectomy), as the negative control; normal (positive control); OVX + OVA (ovariectomy and transplantation) (control), treated with 0.5 mL of normal saline; OVX + OVA + 10 mg/kg of rutin; OVX + OVA + 30 mg/kg of rutin; OVX + OVA + 60 mg/kg of rutin; OVX + OVA + 100 mg/kg of rutin; and the autograft. Groups were treated daily. Fourteen days after transplantation, ovarian grafts were collected and processed histologically for follicle number counting. Serum estrogen (E2) and progesterone (P4) levels were evaluated. Furthermore, the expression of Estrogen Receptor alpha (ERα), Estrogen Receptor beta (ERβ), and Progesterone Receptor (PR) in the uterine endometrial tissue was tested using qRT-PCR and western blotting.

Results: A decrease in the number of mature follicles and increase in the number of atretic follicles (mean ± SD: OVX + OVA + 30 = 19.00 ± 1.000, OVX + OVA + 60 = 25.00 ± 5.000, and OVX + OVA + 100 = 23.00 ± 2.646) were observed in all groups treated with rutin in comparison with the control group (mean ± SD: 12.33 ± 2.517) (P value < 0.05). The level of E2 and P4 (mean ± SD: OVX + OVA + 100 = 6.133 ± 1.026) increased in comparison with the OVX + OVA group (mean ± SD: 0.4667 ± 0.2517) (P value < 0.05). The protein expression of ERα (mean ± SD: OVX + OVA + 10 = 1.615 ± 0.1701 and OVX + OVA + 30 = 1.744 ± 0.1779) in comparison with the control group (mean ± SD: 0.7089 ± 0.1131), and ERβ (mean ± SD: OVX + OVA + 10 = 0.7747 ± 0.4365, OVX + OVA + 30 = 0.9220 ± 0.1245, OVX + OVA + 60 = 0.7701 ± 0.2150, and OVX + OVA + 100 = 0.6676 ± 0.1547) increased in a dose-dependent manner in all groups treated with rutin in comparison with the OVX + OVA group (mean ± SD: 0.1534 ± 0.06109) (P value < 0.05). No significant changes in PR were found in groups treated with rutin in comparison with the control group.

Conclusions: The results of the present study indicated that rutin increases E2 and P4 levels in ovarian hetero allograft mice. Rutin also upregulated the expression of ERα and ERβ but had no significant effect on PR.

Keywords

Allografts Estrogen Receptor Alpha Estrogen Receptor Beta Follicle Mice Ovary Polymerase Chain Reaction Progesterone Rutin Transplantation Up-Regulation Western Blot

Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited

1. Background

The risk of sterility is very serious in young women with cancer. Upon the progress in treatments, there are now several options available for fertility preservation, and thus the use of transplantation in reproductive medicine has been considered. Despite being an attractive and helpful method in reproductive medicine in the early days, there are some problems with ovarian transplantation (1). The most important challenge in ovarian tissue transplantation is the survival rate and satisfaction of the functional longevity of the transplanted ovary (2).

During the engraftment and neovascularization of the transplanted ovarian tissue, an initial ischemia usually occurs, followed by a reperfusion period, which leads to the production of reactive oxygen species (ROS) with oxidative properties (3, 4). Superoxide and hydroxyl radicals are involved in tissue injury through the initiation of lipid peroxidation and damage the cell membrane in the transplanted tissue (5). ROS products are also directly responsible for the oxidative damage of cellular structures, such as DNA, RNA, proteins, and lipid ingredients in ischemic tissues (6). The mentioned events can lead to follicular loss and engraftment insufficiency in the transplanted ovarian tissue (7-9). Due to the critical effect of oxidative stress in transplantation, as described above, antioxidants can be applied to reduce the production of free radicals. Such drugs with antioxidant properties are able to counter the consequences of ischemia and reperfusion injury after ovarian tissue transplantation.

Rutin (quercetin-3-rhamnosyl-glucoside) is a flavonol, abundantly found in apples, tea, onions, and other plants (10, 11). It has multiple pharmacological activities, including antibacterial (12), antitumor, antiulcer (13), myocardial-protecting (14), vasoprotective, immunomodulatory, antioxidant, cytoprotective, and neuroprotective activities (15). Moreover, in the reproductive system, rutin has shown a possible protection of the testicular tissue and reproduction from oxidative stress in diabetes mellitus (16) and has led to the amelioration of cisplatin-induced reproductive toxicity (17). Considerable interest has recently been directed to the role and usage of natural antioxidants as a means of preventing oxidative damage in different conditions with a high oxidative stress (13).

Steroids play a prominent role in the acceptance of transplanted tissues. Extensive evidence suggests that estrogen directly modulates angiogenesis via effects on endothelial cells (18). Estrogen, 17β-estradiol (E2), plays an important role in regulating the proliferation and maturation of ovarian follicles. It has a critical function in the female reproductive system (19, 20). Its effects are mediated through two types of receptor: Estrogen Receptor alpha (ERα) and Estrogen Receptor beta (ERβ). These nuclear receptors mediate the biological effects of estrogens and anti-estrogens and act mainly in the regulation of the Estrogen Receptor (ER) target-gene expression (21). Progesterone (P4) is an endogenous steroid involved in the menstrual cycle and pregnancy (22). The physiological effects of P4 are exerted by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors (23).

2. Objectives

The above-mentioned facts form the basis for studying whether the antioxidant mechanisms involved in rutin-mediated protection from ischemic damage after ovarian transplantation can improve the conditions of fertility. Consequently, the present animal experimental study aimed to investigate the effects of different concentrations of rutin on the return of fertility after ovarian heterotopic allograft and the expression of ERα, ERβ, and PR in Balb/c mice.

3. Methods

3.1. Animals

The present animal experimental study was conducted on 40 adult female Balb/c mice, 5 - 6 weeks of age, weighing approximately 25 - 35 g. Animals were obtained from the Animal House of Jundishapur University of Medical Sciences, Ahvaz, Iran, and maintained at standard conditions (temperature 22 - 24°C and humidity 55% - 65% in a 12-hour light-dark cycle) during 2016 - 2018 in Jundishapur University of Medical Sciences, Ahvaz, Iran. Animals had free access to sufficient amounts of water and food during the study. This study was performed in accordance with the guidelines for Animal Research of the National Institutes of Health (NIH, Bethesda, MD, USA). The ethical code was approved by the Ethics Committee of Jundishapur University of Medical Sciences, Ahvaz, Iran, on 16 May, 2017 (Code: IR.AJUMS.REC.1396.40). For mating, a male and a female animal were housed in plastic cages. Ten days after birth, the newborn female mice were selected for the donation of the ovary to adult female mice. According to a similar study by Domitrovic et al. (24) in 2012, the mean relative weight of the liver in the control group was 5.0 ± 7.4 and, in the intervention group with CCl4, it was 4.0 ± 7.6. By considering the confidence level of 95% and a test power of 80%, five mice for each study group were estimated using the following formula.

The animals were divided into eight groups by permuted block randomization. The eight groups consisted of OVX (ovariectomy/negative control), undergoing ovariectomy; the normal group (positive control), normal and untreated mice; OVX + OVA (ovariectomy and transplantation) (control), undergoing ovariectomy and transplantation, treated with 0.5 mL of normal saline; OVX + OVA + Rut 10, OVX + OVA + Rut 30, OVX + OVA + Rut 60, and OVX + OVA + Rut 100, undergoing ovariectomy and transplantation, treated with 10, 30, 60, and 100 mg/kg of rutin, respectively; and autograft, without any treatment. All groups were treated once a day for two weeks with the intraperitoneal injection of normal saline for the control group and rutin for other groups. Rutin was purchased from the Sigma Aldrich Company (St. Louis, MO) and dissolved in DMSO. All mice were anesthetized using ketamine (60 mg/kg) and xylazine (6 mg/kg) and then ovariectomized in sterile conditions. One week after ovariectomy, two ovaries were transplanted under the muscles behind the neck.

3.2. Sample Collection

After transplantation, to ensure that the estrus phase is resumed in the OVX + OVA mice, the vaginal smear was prepared every day, 3 - 4 days after transplantation. For serum collection, 24h after the last dose of rutin, 1.5 mL of blood was collected from the heart. The blood sample was centrifuged at 3000 g for 5 min, and the serum was separated. Then, all samples were kept at -20 °C until use for the hormonal assay.

3.3. Hormonal Assay

Serum E2 and P4 levels were analyzed by the ELISA method in duplicate using a progesterone kit (Crystal Chem, USA) with the sensitivity of 0.04 ng/mL and assay range of 0.4 - 100 ng/mL; and an estrogen kit (Cusabio, USA) with the sensitivity of 40 pg/mL and assay range of 40 - 1500 pg/mL, according to the manufacturer's instructions.

3.4. Histology

Two weeks after transplantation, all grafted ovaries were collected and fixed in 10% formalin and then the tissues were embedded in paraffin, serially sectioned at 5 µm, and stained with the Haematoxylin and Eosin. Ovaries of age-matched 32-day-old mice were histologically processed as described above as the in-vivo development of normal control. Microphotography slides were obtained using a light microscope (Olympus CH-BI45-2) with the magnification of 20X and 40X and assessed by three experts. Primordial, primary, preantral, antral and atretic follicles were counted.

3.5. qRT-PCR

Total RNA was extracted from endometrial tissue using RiboEx (GeneAll Biotechnology Co, USA) according to the protocol of the manufacturer. Extracted RNA was kept at -80°C. For cDNA synthesis, 500 ng of total RNA was added to the cDNA synthesis tube (Thermo Scientific, USA). The program of cDNA synthesis was: 30°C for 5 min, 42°C for 1 h, 70°C for 5 min, heated at 95°C for 5 min, and stored at -20°C.

qRT-PCR primers were designed using AlleleID 6.0 software and ordered to the CinnaGen Company (Tehran, Iran) for commercial synthesis. The primer sequences of the gene of interest are fully described in Table 1. qRT-PCR reactions were performed in the ABI StepOne instrument (Applied Biosystems, USA) under the following conditions: 95°C for 15 s and 60°C for 1 min for up to 40 cycles. The data are presented as the relative quantity of normalized-target RNA using the ∆∆CT method (25) (internal control gene GAPDH). Each sample was examined in duplicate.

Table 1. List of the Primer Sequences of Interest Gene
GenePrimers
GAPDH
FW5’-AGCAAGGACACTGAGCAAGAG-3’
RE5’-GGATGGAAATTGTGAGGGAGATG-3’
ERα
FW5’-TAGCGGCAACAGTGAAATCC-3’
RE5’-TGGCAAGGTAAGCAATGGC-3’
ERβ
FW5’-ATGGACTGTAGAACGGTGTGG-3’
RE5’-GTGAGGTAGGAATGCGAAACG-3’
PR
FW5’-GATTCAGAAGCCAGCCAGAG-3’
RE5’-CACAGGTAAGCACGCCATAG-3’

3.6. Western Blotting

The expression of ERα, ERβ, and PR was assessed by the Western blot technique. It was prepared using the lysis buffer containing the RIPA buffer and a protease inhibitor cocktail. Total protein was measured by the Bradford method. Equal amounts of protein were resolved by 12% SDS-polyacrylamide gel electrophoresis using the Bio-Rad system (Mini-PROTEAN Tetra System, USA), and then transferred on the PVDF membrane (Immobilon-p transfer membranes) using the wet transfer method. The membrane was blocked overnight with casein 1% in Tris Buffered Saline (TBS) with Tween-20. The membrane was probed with primary antibody (Abcam, USA) and then with horseradish peroxidase-conjugated secondary antibody (Abcam, USA). Finally, ECL plus was used for the final detection of protein bands, and all data were analyzed using the ImageJ software.

3.7. Statistical Analysis

The data were analyzed using IBM SPSS Statistic Software for Windows, version 22.0 (IBM Corp., Armonk, N.Y., USA). All devices and equipment were calibrated to obtain unbiased data during the study. In this study, descriptive statistics, including mean, standard deviation, and error bar were used to plot the mean and standard deviation of each group. Descriptive data are presented as a mean ± standard deviation. The normality of variables was examined using the Shapiro-Wilk test (P value > 0.05). Since the data showed a normal distribution, one-way ANOVA with a post-hoc test was used. In this study, to determine the agreement between the two observers, the kappa coefficient was calculated which was 0.80, indicating that the agreement rate is reasonable. The P value < 0.05 was considered statistically significant.

4. Results

4.1. Level of E2 and P4

As illustrated in Figure 1, the serum level of E2 and P4 dramatically decreased in the OVX group in comparison with the control group. In OVX, OVX + OVA , OVX + OVA Rut 10, OVX + OVA Rut 30, OVX + OVA Rut 60, OVX + OVA Rut 100, autograft and normal groups, the level of E2 was, 0, 25, 32.6, 31, 21.2, 23.3, 28, 26.3 (pg/mL) respectively and the level of P4 was 0.4, 0.4, 2.9, 2.93, 1.26, 6.13,6.5, 8 (ng/mL), respectively. The level of E2 increased in all OVA + rutin groups and the autograft group in comparison with the OVX group (P value < 0.01). Intra-group analysis of the relation between various concentrations of rutin showed the most increase in 0, 10, 30, 100, and the autograft groups. As expected, the level of P4 was increased in all OVA + rutin groups, in addition to the autograft group, compared to the OVX + OVA group. Intra-group analysis indicated the less effect of OVX + rutin 60 compared to OVX + rutin 0, 10, 30, 100, and also the autograft group (P value < 0.0001) (Table 2).

Serum ELISA results for estrogen and progesterone
Figure 1. Serum ELISA results for estrogen and progesterone
Table 2. Changes in Sex Hormones in Groups Receiving Rutina
GroupsEstrogen, pg/mLProgestron, ng/mL
OVX0.0 ± 0.0b0.4667 ± 0.1155
Normal26.33 ± 4.7268.000 ± 1.600b
Treat 025.00 ± 10.150.4667 ± 0.2517
Treat 1032.67 ± 4.1632.900 ± 0.5196
Treat 3031.00 ± 2.0002.933 ± 0.6429
Treat 6021.23 ± 14.031.267 ± 0.6658
Treat 10023.33 ± 0.57746.133 ± 1.026b
Autograft28.00 ± 6.2456.500 ± 2.166b

aValues are expressed as mean ± SD.

bSignificant difference compared to treat 0 (P < 0.05).

4.2. Follicle Development

The presence of numerous cysts in all groups under treatment with rutin was observed. The number of these cysts was decreased with a decrease in the dose of rutin. The number of primordial follicles in treated groups was in the following order: treat 60 > treat 0 > treat 30 > treat 10 > treat 100. Moreover, the number of primary follicles in treated groups was in the following order: treat 60 > treat 10 > treat 30 > treat 100 > treat 0. Also, the number of preantral follicles in the treated groups was in the following order: treat 10 > treat 60 > treat 30 > treat 0 > treat 100 (P value < 0.0001). In addition, the number of antral follicles in the treated groups was in the following order: treat 10 > treat 30 > treat 60 > treat 0 > treat 100 (P value < 0.0001). Finally, the number of atretic follicles in the treated groups was in the following order: treat 60 > treat 100 > treat 30 > treat 10 = treat 0 (P value < 0.001) (Figure 2 and Table 3).

Number of follicles in examining the ovaries in normal, OVX + OVA, OVX + OVA + Rut 10, OVX + OVA + Rut 30, OVX + OVA + Rut 60, and OVX + OVA + Rut 100 groups; (A) number of primordial follicles, (B) number of primary follicles, (C) number of preantral follicles, (D) number of antral follicles, and (E) number of atretic follicles.
Figure 2. Number of follicles in examining the ovaries in normal, OVX + OVA, OVX + OVA + Rut 10, OVX + OVA + Rut 30, OVX + OVA + Rut 60, and OVX + OVA + Rut 100 groups; (A) number of primordial follicles, (B) number of primary follicles, (C) number of preantral follicles, (D) number of antral follicles, and (E) number of atretic follicles.
Table 3. Follicular Density in Grafted Ovariesa
GroupsNo. of Follicles
PrimordialPrimaryPreantralAntralAtretic
Normal19.67 ± 5.68619.67 ± 5.68621.00 ± 1.000b23.00 ± 1.000b5.000 ± 1.000b
Treat 017.67 ± 2.51716.67 ± 2.0820.3333 ± 0.57740.3333 ± 0.577412.33 ± 2.517
Treat 1012.33 ± 2.51717.67 ± 2.51713.00 ± 1.000b10.67 ± 3.055b12.33 ± 2.517
Treat 3013.67 ± 3.78617.33 ± 2.0821.000 ± 1.0001.667 ± 1.52819.00 ± 1.000b
Treat 6019.67 ± 1.52819.67 ± 1.5283.000 ± 1.000b1.000 ± 1.00025.00 ± 5.000b
Treat 10012.00 ± 3.00017.00 ± 4.3590.0 ± 0.00.0 ± 0.023.00 ± 2.646b

aValues are expressed as mean ± SD.

bSignificant difference compared to treat 0 (P < 0.05).

4.3. PR, ERα, and ERβ mRNA Expression

Results indicated that, as expected, the PR, ERα, and ERβ mRNA expression in the OVX group was almost zero. OVX + rutin 10 and 30 groups had the highest levels of ERs mRNA (P value < 0.001). The mean mRNA expression level of ERα was 0.3513 ± 0.5687, 1.000 ± 0.0, 5.430 ± 1.000, 5.218 ± 1.000, 2.250 ± 1.000, 2.143 ± 1.100, 5.429 ± 1.000 and 5.760 ± 1.000 (Figure 3A); The mean mRNA expression level of ERβ was 0.3344 ± 0.1000, 1.000 ± 0.0, 3.608 ± 0.5774, 4.497 ± 1.000, 2.877 ± 1.528, 3.442 ± 1.000, 0.4454 ± 0.5774 and 2.998 ± 1.000 (Figure 3B) and The mean mRNA expression level of PR was 0.6776 ± 0.5774, 1.000 ± 0.0, 1.073 ± 1.000, 1.101 ± 1.000, 1.733 ± 1.000, 1.018 ± 1.000, 1.038 ± 1.000 and 3.528 ± 1.000 in OVX, OVX + OVA, OVX + OVA Rut 10, OVX + OVA Rut 30, OVX + OVA Rut 60, OVX + OVA Rut 100, autograft and normal, respectively. In all the treated groups, an increase in PR mRNA versus the OVX group was shown, but there was no statistically significant difference (Figure 3C).

mRNA expression level of ERα, ERβ, and PR (A, B, C); protein expression level of ERα, ERβ, and PR (D, E, F, G)
Figure 3. mRNA expression level of ERα, ERβ, and PR (A, B, C); protein expression level of ERα, ERβ, and PR (D, E, F, G)

4.4. Protein Expression Level of PR, ERα, and ERβ

The protein expression level of PR, ERα, and ERβ within the endometrial tissue of the mice had the same pattern across groups. In OVX, OVX + OVA, OVX + OVA Rut 10, OVX + OVA Rut 30, OVX + OVA Rut 60, OVX + OVA Rut 100, autograft and normal, the level of ERα was, 0.0044, 0.81, 1.88, 1.78, 1.46, 0.56, 1.76, 1.65, respectively. The level of ERβ was 0.025, 0.14, 1.25, 1.05, 0.56, 0.80, 0.19, 0.50, respectively, and the level of PR was 0.09, 0.21, 0.14, 0.88, 0.10, 0.28, 0.09, and 0.59 respectively. The rates in the OVX + rutin 100 and 0 groups had the lowest levels of ERs (P value < 0.05). The highest level of ERα and ERβ was seen in OVX + rutin 10 and 30. There were no detectable expressions in the OVX group (P value < 0.01). Moreover, the protein expression of PR in all OVX + OVA groups treated with rutin was increased in comparison with the OVX group, but there was no significant difference (Figures 3D - 3G and Table 4).

Table 4. Protein Expression Level of Estrogen Receptor Alpha (ERα), Estrogen Receptor Beta (ERβ), and Progesterone Receptor (PR)a
GroupsERaERbPR
OVX0.007864 ± 0.0038370.05174 ± 0.037780.1156 ± 0.06584
Normal1.495 ± 0.3662b0.6670 ± 0.1752b0.6010 ± 0.1626b
Treat 00.7089 ± 0.11310.1534 ± 0.061090.1423 ± 0.09290
Treat 101.615 ± 0.1701b0.7747 ± 0.4365b0.1593 ± 0.06674
Treat 301.744 ± 0.1779b0.9220 ± 0.1245b0.2064 ± 0.1529
Treat 600.9776 ± 0.76170.7701 ± 0.2150b0.1591 ± 0.06259
Treat 1000.4953 ± 0.20980.6676 ± 0.1547b0.2763 ± 0.05559
Autograft1.770 ± 0.1000b0.1860 ± 0.027880.2537 ± 0.1264

aValues are expressed as mean ± SD.

bSignificant difference compared to treat 0 (P < 0.05).

5. Discussion

Results of the present study demonstrate that fresh hetero allograft ovarian transplantation with different concentrations of rutin in mice can restore ovarian function, as evidenced by the production of E2 and P4 along with the expression of ERα, ERβ, and PR in the transplanted mice. Also, results showed that the use of rutin in allograft for restoring the ovarian function could have an equal value to autograft transplantation in some concentrations.

Beazley and Nurminskaya explored the effects of flavonoid on fertility in female mice. They demonstrated that quercetin leads to a 60% reduction in the number of litters, but enhances folliculogenesis in the ovaries of female offspring (26). In this study, consistent with the study by Beazley and Nurminskaya, rutin as a flavonoid caused an increase in the maturation of follicles but was not able to increase fertility (increased atretic follicles).

Treatment of OVX + OVA mice with rutin possibly restored the estrous cyclicity. However, the rutin-treated groups at different concentrations showed different serum levels of E2 and P4.

In the present study, E2 concentration in plasma was significantly increased. Guo et al. in a similar study reported that the administration of rutin has an effect on ovariectomized rats similar to the administration of E2. They concluded that rutin is able to increase E2 concentration in serum and mammary glands (27). In another study on flavonoids, Tan et al. demonstrated the same role of these molecules in increasing E2 levels in pubertal female rats (28). In addition, Nynca et al. reported genistein action (a flavonoid) on E2 production by porcine granulosa cells of medium follicles. All doses of genistein increased basal E2 secretion by the granulosa cells cultured for 48 h (29). In contrast, daidzein as another flavonoid did not alter the granulosal secretion of E2 (29).

P4 was also studied as a hormone with fluctuations in ovary activity and follicle growth. Interestingly, the level of P4 was higher in the autograft group than rutin10, 30, and 60 groups, while there was no significant difference in comparison with rutin 100. Jahan et al. in a study on rats with polycystic ovary syndrome demonstrated an increase in P4 due to the administration of rutin at two doses of 100 mg/kg and 150 mg/kg (30). In contrast, in other studies, genistein inhibited P4 production by the porcine granulosa cells of medium follicles (29) and porcine luteinized granulosa cells isolated from large follicles (31).

Results showed that ERβ expression in the endometrial tissue was significantly increased in all OVX + OVA groups treated with rutin versus OVX and treat 0 groups. As demonstrated in the E2 pattern, an increase in ERβ in the treated group had a significantly higher protein expression level than the autograft group, suggesting that rutin increased ERα expression in the endometrial tissue. The protein expression level of ERα has the same pattern as ERβ in all treated groups in comparison with control, OVX, and OVX + OVA groups. A prominent difference was found in the expression level of ERα which was considerably higher in the autograft group in comparison with all other groups. In their study on the possible pathway using which rutin can ameliorate oxidative injury, Hong et al. report that rutin can attenuate the ischemia/reperfusion injury in ovariectomized rats via ER-mediated signaling pathways (BDNF-TrkB and NGF-TrkA signaling pathway) (32). The structural similarity of rutin to endogenous estrogen and plant estrogen makes this molecule able to be absorbed by target cells, be bound to ER, and then exert estrogen-like effects (33). In addition to the direct effects of rutin on ER activation and signaling initiation, this agent is able to increase the production of estrogen directly, as mentioned by Guo (27). Similarly, Nynca et al. demonstrated that genistein caused a significant increase in the ERβ mRNA level in granulosa cells of large follicles (31) and granulosa cells of medium follicles (29). However, it did not alter ERα mRNA level in the culture porcine granulosa cells (29, 31). In contrast, the expression of ERβ protein was affected by genistein in the granulosa cells of medium follicles (29), but it was not detected in the granulosa cells of large follicles (31). Daidzein decreased mRNA expression of ERα in medium follicles, but the expression of ERβ mRNA was not affected by daidzein. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein upregulated the expression of ERβ protein in the cells (29).

The PR expression in the endometrial tissue had a decreased pattern in comparison with the control group. The maximum protein expression level of PR was observed in the treat rutin 100 group. In all transplanted groups, an increase of PR versus the OVX group was shown. However, in groups 0, 10, 30, 60, and the autograft group, there was no statistically significant difference. These results suggest that rutin is able to enhance PR. Parallel with this study; Rosenberg et al. demonstrated that flavonoids have a progesterone-like antagonist activity. In their study on PR in breast cancer cell lines, it was shown that flavonoids bind to the PR and act as a blocker. They demonstrated that flavonoids could modulate PR expression (34). It seems that rutin, as a flavonoid component, has a modulatory effect on PR beside its antioxidant and estrogen-like abilities. In agreement with Rosenberg, no statistically significant change in PR was observed in the present study.

5.1. Strengths and Limitations

5.1.1. Limitations

- The sample size was small (n = 5) for each group.

- No additional evaluation methods such as immunohistochemistry were used.

5.1.2. Strengths

- Evaluation methods such as qRT-PCR and Western blotting were employed.

- Four dosages of rutin were tested.

5.2. Conclusions

The present study demonstrates that the effects of rutin on restoring the estrous cyclicity after transplantation rely on its antioxidant effect on the inhibition of the increased oxidative stress. The results of the present study indicated that rutin increased the E2 and P4 levels in ovarian hetero allograft mice. Rutin also upregulated the expression of ERα and ERβ but had no significant effect on PR. ER upregulation led to an enhanced function of estrogen, improved the engraftment and function of the transplanted ovarian tissue, and restored estrous cyclicity.

Footnotes

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