Modulation of TGF-β and FOXL2 Pathways by Genistein and Ethinyl Estradiol in Radiation- Induced Ovarian Toxicity in Wistar Rats

Radiotherapy (RT) represents a ubiquitous therapeutic modality in cancer management, with approximately eighty percent of neoplastic patients undergoing RT sessions during the course of their treatment [1]. Unfortunately, RT is associated with the undesirable outcome of infertility or Premature Ovarian Failure (PMOF), particularly in the treatment of cervical and rectal neoplasms [2]. The radiotherapy sessions induce the generation of Reactive Oxygen Species (ROS), which, coupled with a reduction in antioxidant pathways in healthy cells, damages them [3]. In the ovarian milieu, this oxidative-antioxidative disequilibrium, favoring the former, is posited as a plausible rationale for RT-induced PMOF, primarily impacting ovarian germ cells [4].

The ROS production attendant to RT can activate cellular apoptotic cascades, instigating DNA damage [5] and mitochondrial dysfunctions mediated by the activation of tumor protein p53. This cascade heightens the permeability of the mitochondrial outer membrane, leading to the excessive production of Cytochrome-c (Cyt-c) and caspases [6]. Numerous investigations underscore the pivotal role of apoptosis [7,8] and the augmented production of transforming growth factor beta isoforms - TGFB-1, TGFB-2, TGFB-3 [9] in RTAssociated Oocyte Damage (RAOD) and loss [10]. To alleviate RAOD, the development of therapeutic agents capable of interrupting oxidative stress and apoptosis pathways is imperative.

Genistein (GEN), derived from soybeans [11], shares structural homology with human estrogen and is thus classified as a phytoestrogen that selectively targets estrogen receptors -beta (ER-β) [12]. It functions as a selective estrogen receptor modulator (SERM) [12] and manifests radioprotective potential in gastrointestinal [13], pulmonary [14] and testicular domains [15], attributable to its antioxidant and anti-fibrotic attributes [16-19]. Previous reports underscore the potential of GEN in safeguarding ovaries against carcinogenesis [20] and prolonging ovarian lifespan [21,22]. The primary objective of the present study is to scrutinize the capacity of GEN to ameliorate RT-associated Ovarian Failure (OF) in albino Wistar rats, alongside an exploration of the underlying molecular pathways.

Chemicals

The AMH ELISA assay, ELISA kits, and Glutathione Peroxidase (GPx) kits were procured from Sino Pharm Chemical Reagent Co., Ltd. (China). Genistein (GEN), ethinyl Estradiol (E2), and di-methylsulfoxide were acquired from Sigma Chemical Co. (USA).

Animals

One hundred albino Wistar rats were utilized with an average weight of 30±10 gm and an average age of 3 weeks. The rats were individually housed and provided free access to chow (El104 Nasr Pharmaceutical Company, Egypt) and water. Daily records were maintained for food, water consumption and any signs of clinical morbidity. The light: dark cycle adhered to a 12:12 regimen, and the temperature was maintained at a constant 25 °C.

Study design

The rats were randomly allocated into five groups (n=20). The phases of the estrous cycle were ascertained through a fourteenday daily analysis of vaginal secretions to ensure cycle regularity, with only female rats displaying consistent phases being included [23]. The groups comprised the Control group (C-group), receiving 3ml/kg of vehicle (V) [di-methyl-sulfoxide and corn oil (1:10)] OD for ten days; I-group, receiving 3ml/kg of V, OD for ten days, with a single dose of 3.4 gray γ-rays administered on day-7 to induce Ovarian Failure (OF); GEN-group, receiving GEN (6mg/kg B.W, Intraperitoneal Injection (IP), OD for ten days; GEN/I group, receiving GEN (6mg/kg B.W, IP, OD for ten days ), with a single dose of 3.4 gray γ-rays administered on day-7; E2/I-group, receiving E2 (0.2mg/kg B.W., Subcutaneous Injection (SC), OD for ten days), with a single dose of 3.4 gray γ-rays administered on day-7.

Rat’s total body weight and on day10, blood was collected from the tail vein, centrifuged and the resultant serum was stored at -80 °C. Upon study completion, rats were euthanized with sodium pentobarbital (intraperitoneal injection, 60mg/kg b.w.). Ovaries were meticulously dissected, weighed and subjected to subsequent biochemical and histopathological examinations.

Serum biochemical analysis

The quantification of serum Anti-Müllerian Hormone (AMH) levels was conducted using the ELISA Kit (CSB-E11162r) (LSBio, USA), while serum E2 levels were determined using commercial kits (RTC009R) (BioVendor, USA).

Histopathological examination

Hematoxylin and Eosin (H&E) staining was conducted following Ahmed’s [24] protocol. Fresh ovaries were processed, embedded in paraffin and sectioned into five μm thick sections. Staining was performed with hematoxylin and eosin and Image J 1.24 v. software was employed to analyze thirty fields per section.

Ovarian homogenate biochemical analysis

Ovaries were homogenized, and GSH levels were determined according to Beutler et al. [25], while GPx activity was assessed following the protocol described by Paglia & Valentine [26].

Real-time Polymerase Chain Reaction (PCR)

Quantification of BAX, TGF-β mRNA, ER-β, Bcl-2, and Forkhead box protein L2 (FOXL-2) levels was performed using realtime PCR (Model: 7900HT) (Thermo Fisher, USA). Ovary tissue samples were lysed with the SE-Quoia Kit (Bio-Rad, USA), and RNA to cDNA transcription was executed. PCR conditions included an initial denaturation at 96 °C (four min.), followed by 40 cycles of 96 °C (twenty sec.), 63 °C (thirty sec.) and 72 °C (thirty sec.). Primer sequences for each gene were summarized in Table 1.

Table 1:List of primers sequences used for the analysis of gene expression.

Immunohistochemistry examinations

Immunohistochemistry was conducted as per Young and Morrison [27]. Paraffin-embedded tissue sections were sliced (five μm thick) and mounted on charged slides. Sections were subjected to antibody treatment and stained with DAB. Image J 1.24 v. was used to analyze ten fields per section [% proliferating cells = number of PCNA immune-positive granulosa cells divided by total number of granulosa cells].

Statistical analysis was performed using Statistical Package for Social Sciences V. 23 (SPSS Inc., USA). Post hoc Tukey-Kramer test was employed for group comparisons, and data were expressed as mean ± standard deviation. The statistical significance of differences between groups was validated using one-way analysis of variance (ANOVA), with a probability value considered significant if <0.05.

Effect of GEN and E2 administration on ovarian and total body weights following whole-body irradiation

A conspicuous reduction in both ovarian and total body weights was evident in the I-group, exhibiting a decrement of 20% and 13%, respectively, compared to the C-group. The GEN/I, E2/I, and GEN groups displayed a non-significant decline in both ovarian and total body weights relative to the C-group (Figure 1).

Figure 1:Effect of GEN and E2 administration on total body weight change (A) and relative ovarian weights (B) after whole-body irradiation. I-group shows a significant (p <0.05) decrease of both ovarian and total body weight, while GEN/I, GEN in addition to E2/I groups show a non-significant difference of same parameters as compared to C-group. *Significant (p <0.05) difference in comparison to C-group. Data are presented as mean ±SD, (n=20).

Effect of GEN and E2 administration on serum hormones following whole-body irradiation:

The I-group manifested a notable reduction in serum Anti- Müllerian Hormone (AMH) and Estradiol (E2) concentrations by 66% and 45%, respectively, in comparison to the C-group. The GEN/I and E2/I group significantly elevated serum AMH and E2 levels, restoring them to C-group levels. The GEN-group demonstrated no significant deviation in serum AMH and E2 levels relative to the C-group (Figure 2).

Figure 2:Effect of GEN and E2 administration on serum AMH (A) and E2 (B) levels after whole-body irradiation. I-group shows a significant (p < 0.05) reduction in serum AMH and E2 concentrations while GEN/I and E2/I groups show a significant (p < 0.05) increase in serum levels comparable to C-group levels. GEN-group shows a nonsignificant difference in same hormones as compared to C-group. *Significant (p < 0.05) difference in comparison to C-group. Data are presented as mean ±SD, (n=20).

Effect of GEN and E2 administration on histological architecture of ovaries following whole-body irradiation:

Histological scrutiny disclosed typical ovarian architecture in the C and GEN groups, characterized by various maturation stages of ovarian follicles within the ovarian cortex periphery. The I-group exhibited hemorrhagic areas, inflammatory cell infiltration, and interstitial fibrosis, concomitant with a significant reduction in healthy primordial, preantral, and antral follicles, and a significant increase in atretic follicles compared to the C-group. The GEN/I and E2/I group showcased the restoration of healthy ovarian follicles, with the GEN/I group exhibiting a noteworthy increase in all follicle types and a reduction in atretic follicles relative to the I-group. Intriguingly, the E2/I group demonstrated a significant increase in the primordial follicle population compared to the GEN/I group and a non-significant increase in other follicle types (preantral, antral, and atretic) relative to the I-group, alongside a significant rise in primordial follicles compared to the GEN/I group (Figure 3).

Figure 3:(A-E) Photomicrograph of tissues of the ovary stained with hematoxylin and eosin (X 400), (n=20). C-group (A) and GEN-group (D) show healthy ovarian follicles at different stages of maturation. Sections of I-group (B) shows multiple hemorrhagic areas with inflammatory cells infiltration. Few numbers of ovarian follicles are replaced by atretic ones. GEN-group (C), GEN/I-group (D) and E2/I group (E) show normal histological architecture. (F) Morphometric analysis of ovarian follicles. I-group shows a significant (p < 0.05) reduction of healthy follicles in addition to a significant (p < 0.05) increase of atretic follicles numbers as compared to C-group. Significant (p < 0.05) increase of all ovarian follicles’ population in GEN/I group with a significant (p < 0.05) decrease of atretic ones as compared to I-group is noticed. E2/I group shows a significant (p < 0.05) increase of primordial follicles population as compared to I-group in addition to a significant (p < 0.05) increase of primordial follicles numbers as compared to GEN/I group. *Significant (p < 0.05) difference in comparison to C-group. #Significant (p < 0.05) difference in comparison to I-group. Data are presented as mean ±SD, (n=20).

Effect of GEN and E2 administration on reduced GSH and GPX levels in ovarian homogenate following whole-body irradiation:

The I-group exhibited a significant reduction in glutathione (GSH) and GPx levels by 68% and 41%, respectively, in comparison to the C-group. Conversely, the GEN/I, E2/I, and GEN groups demonstrated a non-significant reduction in GPx and reduced GSH levels relative to the C-group (Figure 4).

Figure 4:Effect of GEN and E2 administration on reduced GSH (A) and GPx (B) in ovarian homogenate after whole-body irradiation. I-group shows a significant (p < 0.05) reduction in GSH and GPx levels (by 68% and 41%, respectively) as compared to the C-group. *Significant (p < 0.05) difference in comparison to C-group. Data are presented as mean ±SD, (n=20).

Effect of GEN and E2 administration on bcl-2 and bax expressions following whole-body irradiation:

The I-group manifested a significant increase in Bax gene expression (by 289%), an elevation in the Bax/Bcl-2 ratio (by 248%) and a significant decrease in Bcl-2 gene expression (by 47%) compared to the C-group. The GEN/I-group demonstrated a significant decrease in Bax expression (by 57%), an increase in Bcl- 2 expression (by 91%) and a significant reduction in the Bax/Bcl- 2 ratio (by 62%) relative to the I-group. The E2/I-group displayed no significant difference in Bax expression, a significant increase in Bcl-2 expression and a significant reduction in the Bax/Bcl-2 ratio compared to the I-group. Notably, the E2/I-group exhibited a significant increase in Bax and Bcl-2 expression with no significant difference in the Bax/Bcl-2 ratio relative to the GEN/I-group, which demonstrated no significant difference in Bax and Bcl-2 expression compared to the C-group (Figure 5).

Figure 5:Effect of GEN and E2 administration on Bax (A), Bcl-2 (B) gene expressions and BAX/BCL-2 ratio (C) after whole-body irradiation. I-group shows a significant (p < 0.05) increase in expression of Bax gene and Bax/Bcl-2 ratio as compared to the C-group. GEN/I-group shows a significant (p < 0.05) decrease in expression of Bax and Bax/ Bcl-2 ratio associated with a significant (p < 0.05) increase of Bcl-2 expression if compared to I-group. E2/I-group shows a significant (p < 0.05) increase of Bcl-2 expression with a significant (p < 0.05) reduction of Bax/Bcl-2 ratio if compared to I-group. E2/I -group shows a significant (p < 0.05) increase of Bax and Bcl2 expression if compared to GEN/I-group. *Significant (p < 0.05) difference in comparison to C-group. #significant (p < 0.05) difference in comparison to I-group. Data are presented as mean ±SD, (n=20).

Effect of E2 and GEN administration on gene expressions of ER-β, FOXL-2, and TGF-β following whole-body irradiation:

The I-group demonstrated a significant decrease in FOXL2 and ER-β expression levels (by 0.46-fold and 0.67-fold, respectively) and a significant increase in TGF-β expression (by 285%) compared to the C-group. The GEN/I and E2/I group exhibited a significant increase in ER-β and FOXL-2 expressions relative to the I-group. The GEN/I-group showed a significant decrease in TGF-β expression (by 56%) compared to the I-group. The E2/I-group displayed a significant increase in TGF-β expression compared to the C-group and a significant increase in FOXL-2 expression compared to the C and GEN groups (Figure 6).

Figure 6:Effect of GEN and E2 administration on ER-β (A), FOXL-2 (B) and TGF-β (C) gene expressions after whole-body irradiation. I-group shows a significant (p < 0.05) decrease in FOXL2 and ER-β expressions levels, in addition to a significant (p < 0.05) increase of the expression of TGF-β if compared to C-group, while GEN/I and E2/I groups show a significant (p < 0.05) increase in ER-β and FOXL-2 expressions if compared to the I-group. GEN/Igroup shows a significant (p < 0.05) decrease in TGF-β expression as compared to the I-group. E2/I-group shows a significant (p < 0.05) increase in TGF-β expression if compared to C-group in addition to a significant (p < 0.05) increase in FOXL-2 expression if compared to C and GEN groups. *Significant (p < 0.05) difference in comparison to C-group. #significant (p < 0.05) difference in comparison to I-group. Data are presented as mean ±SD, (n=20).

Effect of GEN and E2 administration on expression of caspase-3 and cytochrome c following whole-body irradiation:

Immunohistochemistry revealed a negative reaction for caspase-3 and cytochrome c in the C and GEN groups, while the I-group exhibited a robust positive reaction, primarily in cells of atretic follicles. Quantitative analysis demonstrated an increase in optical densities of immune-positive cells (by 79% and 57%, respectively) compared to the C-group (Figure 7 & Figure 8).

Figure 7:(A-E) Photomicrograph of tissues of the ovary stained with anti-cytochrome c antibodies (X 400), (n=20). C and GEN groups have a negative reaction. I-group shows a strong positive reaction (in the form of brownish discoloration) for cytochrome c. (F) Immunoreactivity quantification. *Significant (p < 0.05) difference in comparison to C-group. #significant (p < 0.05) difference in comparison to I-group. Data are presented as mean ±SD, (n=20).

Figure 8:(A-E) Photomicrograph of tissues of the ovary stained with anti-caspase-3 (X 400), (n=20). C and GEN groups have a negative reaction. I-group shows a strong positive reaction (in the form of brownish discoloration of cells) for caspase-3 mainly in cells of atretic follicles. (F)Immunoreactivity quantification. *Significant (p < 0.05) difference in comparison to C-group. #significant (p < 0.05) difference in comparison to I-group. Data are presented as mean ±SD, (n=20).

Said et al. [28] meticulously expounded upon the deleterious repercussions of whole-body radiation on the female pelvic organs, particularly the ovaries, culminating in Premature Ovarian Failure (PMOF). Genistein (GEN) has garnered recognition for its radioprotective prowess across diverse organs, such as the liver, intestine, and testis, attributed to mechanisms encompassing augmented cell survival and repopulation [18, 29,30]. Moreover, GEN has been documented to assuage ovarian toxicity induced by cyclophosphamide, attenuating oxidative stress and mitigating inflammation in the ovaries [22]. This study illuminates GEN’s potential to safeguard ovarian function post-radiotherapy, demonstrating efficacy in preserving ovarian histological architecture, maintaining ovarian follicular reserves, conserving Anti-Müllerian Hormone (AMH) and estradiol hormonal levels, enhancing antioxidant pathways, impeding apoptotic pathways, and modulating FOXL-2 and ER-β expression, alongside downregulating TGF-β expression.

The current investigation unveils a profound loss of various ovarian follicle types (primordial, preantral, and antral) in the I-group four days after the radiation session. These findings align with Thompson et al. [31] who observed that the destruction of primordial follicles hastens the onset of PMOF, resulting in female sterility. The observed ovarian follicular atresia in the I-group concurs with the prior report of Adriaens et al. [32]. Notably, the GEN/I-group exhibits a notable preservation of the primordial follicle population and a reduction in atretic follicle numbers, indicative of GEN’s radioprotective potential. This finding aligns with Chen et al. [33] associating GEN’s ability to prolong the ovarian lifespan in young rats with its capacity to impede the development of primordial follicles into growing follicles, consequently augmenting the ovarian reserve of follicles. The E2/I group significantly increased the primordial follicle population, suggesting that E2 possesses a more pronounced inhibitory effect on primordial follicles than GEN. Furthermore, the E2/I-group shows a non-significant difference in the antral or atretic follicles population compared to the I-group, implying that GEN exhibits more radioprotective potential than E2.

The current study observes a significant reduction in E2 in the I-group, potentially attributed to ovarian toxicity, as described by Jarrell et al. [34] and Langan et al. [35] highlighted the role of diminished E2 secretion in activating the transformation of primordial follicles into growing follicles, potentially leading to PMOF due to the depletion of the follicular stock. Gracia et al. [36] proposed that serum AMH could be considered a marker for ovarian follicular reserve, which was found to be decreased in the current study. The GEN/I and E2/I groups exhibit a significant elevation in serum E2 and AMH levels, consistent with the findings of Saleh & Mansour [22], who reported the role of GEN in restoring E2 and AMH levels in rats treated with cyclophosphamide.

Ionizing radiation’s potential for structural and functional tissue distortion is attributed to its role in the overproduction of free radicals, resulting in damage to cellular macromolecules and proteins (41). The current study demonstrates a significant reduction in Glutathione (GSH) and Glutathione Peroxidase (GPx) levels in the ovaries of the I-group. Pankhurst (2017) attributes ovarian follicles’ apoptosis in adult rats to GSH depletion. On the contrary, the antioxidant potential of GEN ameliorates irradiationinduced toxic effects, consistent with the results of Saleh & Mansour [22], who reported GEN’s role in attenuating chemotherapyinduced ovarian toxicity. Thus, GEN ameliorates oxidative stress induced by radiation, akin to E2.

Radiation-induced apoptosis is linked to DNA damage and oxidative stress. According to Cho et al. [37], the apoptotic pathway is initiated at the mitochondrial level, resulting in follicular cell apoptosis due to caspase activation. Gürsoy et al. [38] add that the apoptosis of ovarian follicle cells is determined by the balance between Bax and Bcl-2. In the current study, the I-group exhibits an upregulated Bax expression associated with a downregulated Bcl-2 expression, indicating a disrupted Bax/Bcl-2 ratio and activation of the apoptotic pathway. Moreover, there is an increased expression of caspase-3 and cytochrome c, which is considered the terminal event preceding the death of follicular cells. GEN administration interrupts the irradiation-initiated apoptotic pathway via the up-regulation of Bcl-2 expression, and down-regulation of Bax, caspase-3, and cytochrome c. The study reveals that the GEN/Igroup exhibits a significantly reduced expression of both caspase-3 and cytochrome c compared to the E2/I-group, confirming GEN’s ability to guard against irradiation-associated apoptosis better than E2, consistent with Chi et al. [39].

Proliferating Cell Nuclear Antigen (PCNA) is essential for DNA replication, and its inhibition is linked to irradiation due to its role in Cyclin-dependent Kinase Inhibitor (CKI) activation, resulting in cell cycle arrest at the G1 phase [40]. PCNA has been acknowledged as a marker of follicular growth and proliferation [41], with a dramatic decrease in expression observed in atretic follicles [42]. In the current study, PCNA expression is lower in the I-group than the C-group, suggesting a cessation of cellular proliferation due to apoptosis. Conversely, there is a significant increase in PCNA immunopositive cells in both the GEN/I and E2/I group. These findings agree with Jarić et al. [43], who hypothesized the protective role of GEN on uterine tissue. Lin et al. [44] attributed the proliferative potential of both GEN and E2 to the upregulation of AMH, acting as a guardian against the toxic effect of irradiation (Figure 9).

Figure 9:(A-E) Photomicrograph of tissues of the ovary stained with anti-PCNA (X 400), (n=20). C and GEN groups have a strong positive reaction while I-group show weak reaction. (F) Quantitative analysis shows that PCNA +ve cells percentage reduced by 67% as compared to C-group. On the other hand, GEN/I and E2/I group reveal that 76% of granulosa cells are PCNA +ve. *Significant (p < 0.05) difference in comparison to C-group. #significant (p < 0.05) difference in comparison to I-group. Data are presented as mean ±SD, (n=20).

Phytoestrogens, including GEN, have the potential to act as natural Selective Estrogen Receptor Modulators (SERMs) [11]. Kuiper et al. [45] reported that GEN has a higher affinity to bind to ER-β than ER-α (by 2000%), and Nadal-Serrano et al. [46] attributed GEN’s antioxidant-like activity to its high binding potential with ER- β, with lower affinity to interact with ER-α compared to E2. Hegele- Hartung et al. [47] hypothesized that ER-β is the main receptor in ovarian follicle cells, promoting proliferation and maturation [48]. Adams et al [49] also attributed GEN’s antiapoptotic and antioxidant potential to its high affinity for ER-β. The current study shows an upregulation of ER-β in the GEN/I and GEN/E2 groups, which may attenuate oxidative stress and apoptosis, accompanied by enhanced proliferation of ovarian follicle cells. Yang et al. [50] suggested that the reduction in caspase-3, cytochrome c, and Bax expressions after GEN administration could be attributed to its role in upregulating ER-β, disrupting the apoptotic pathway initiated in the mitochondria.

TGF-β and FOXL-2, modulators of cellular apoptosis and proliferation regulate the maturation of ovarian follicles. According to Oktem and Urman [10], TGF-β activates ovarian follicle development by enhancing the transformation of primary follicles. The role of TGF-β in follicular development has been previously reported [51], while Zheng et al. [52] reported TGF-β’s pro-apoptotic role in ovarian granulosa cells. TGF-β’s role has also been implicated in radiation-induced PMOF [53] and in negatively affecting E2 secretion [52], consistent with the results of the current study.

FOXL-2 is a transcription factor involved in various ovarian development stages Georges et al. [54] suggested the role of FOXL-2 in most ovarian development stages. Additionally, Qin et al. [55] postulated the importance of FOXL-2 in ovarian follicle proliferation and differentiation. Benayoun et al. [56] attributed FOXL-2’s role in preserving ovarian follicular reserves to its inhibitory potential on follicular maturation, thus maintaining primordial follicles in a dormant state. FOXL-2 mutations are commonly associated with PMOF. The downregulation of FOXL-2 observed in the I-group could be considered one of the underlying causes of primordial follicle depletion. These results are consistent with the previous report of Schmidt et al. [57], who associated the depletion of the primordial reserve with the absence of secondary follicles. Therefore, follicular reserve depletion in response to total body radiation could be attributed to the upregulation of TGF-β and downregulation of FOXL-2 [58]. The current study was limited to estimating the potential of GEN in a rodent experimental model, and further studies on different animal species are required for additional clinical application [59-61].

In conclusion, the current study sheds light on the potential of GEN protects the ovaries against radiation-induced injury through various mechanisms, including its antioxidant ability to enhance ovarian follicle cell proliferation (proven superior to E2 potential). Additionally, GEN caused FOXL-2 up-regulation and TGF-β downregulation, constituting a protective effect against PMOF induced by radiation.

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