Detection of c-Abl and mTERT Expression by DMBA-Induced Mouse Model of Premature Ovarian Insufficiency

Detection of c-Abl and mTERT Expression by DMBA-Induced Mouse Model of Premature Ovarian Insufficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detection of c-Abl and mTERT Expression by DMBA-Induced Mouse Model of Premature Ovarian Insufficiency Ecem Yildirim, Tugce Onel, Aylin Yaba This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7571888/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Journal of Molecular Histology → Version 1 posted 11 You are reading this latest preprint version Abstract The polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), is a potent carcinogen induces DNA damage and causes ovotoxicity with depletion of all follicle types in rat and mouse. c-Abl protein tyrosine kinase is activated by DNA double-strand breaks and proteins involved in the repair of lesions function in telomere control. Telomerase is one of the most important factor to limit telomeric shortening and maintain oocytes number in ovarian reserve. We showed before that mTERT associates directly with the c-Abl tyrosine kinase in mouse granulosa cells. Therefore, in the purposed study we aimed to show the mechanism of depletion of follicle reserve and atresia in the DMBA-induced ovotoxicity mouse model. In our study, we focused on the potential roles of c-Abl protein tyrosine kinase and mTERT telomerase catalytic subunit in ovarian follicles after DMBA exposure. We determined that exposure to DMBA resulted in decreased c-Abl tyrosine kinase activity in response to increased telomerase activity in mouse ovary. In conclusion, our results suggested that c-Abl and mTERT may have a functional role in folliculogenesis and mediate rapid depletion of ovarian follicles in DMBA-induced ovotoxicity mechanism. DMBA (7 12-dimethylbenz-[a]anthracene) Ovotoxicity Mouse c-Abl mTERT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 INTRODUCTION The oocyte reserve that occurs in prenatal life in mammals decreases and depletes during postnatal life. During this depletion period, a very small part of the oocytes complete their functional development and ovulate, while thousands are eliminated before ovulation. With this mechanism called follicular atresia, ovarian aging occurs and the primordial follicle pool is depleted (Borman et al 2000 , Broekmans et al 2007 , Hansen et al 2008 , Hoyer & Sipes 1996 ) and this depletion results infertility or Premature Ovarian Infufficiency (POI). Polycyclic aromatic hydrocarbon 7,12-Dimethylbenz[a]anthracene (DMBA) is an environmental carcinogen that triggers many tumors, is a widely studied model carcinogen for the leading to a decrease in ovarian reserve and premature ovarian failure in rodents (Hoyer et al 2009 ). So how can people be exposed to DMBA in daily life? As the source of DMBA, it is stated that smoking, consuming foods exposed to activated carbon while cooking (such as barbecued food) and inhaling exhaust fumes from automobiles (Gelboin 1980 ). In addition to its carcinogenic effect, it is also stated that DMBA disrupts the normal process of folliculogenesis, causing a decrease in the follicle population and causing POI (Mattison & Nightingale 1980 ). Studies have shown that DMBA causes the activation of the proapoptotic Bax protein and Caspase-3 protein in preovulatory follicles, resulting in the apoptosis of granulosa and theca cells (Tsai-Turton et al 2007 ). When smokers and non-smokers were compared, it was determined that early menopause was induced in smokers (Harlow & Signorello 2000 , Jick 1979 , Mattison et al 1983 ). In addition, it has been shown that the offspring of smoking mothers, even if they do not smoke during pregnancy, have problems such as a decrease in oocyte number, abnormal ovarian function, decreased fertility and/or premature menopause (Jurisicova et al 2007 ). In a study with pig oocytes, it was shown that; DMBA triggers changes of meiotic cycle, double strand break, early apoptosis of cumulus cells and histone methylation. At the same time, DMBA causes a significant increase in reactive oxygen species in oocytes both in the presence and absence of cumulus cells around the oocytes (Song et al 2017 ). Abelson Tyrosine Kinase (c-Abl) proto-oncogene is a member of a non-receptor protein tyrosine kinase family that provides its biological effects through kinase activity (Zhu & Shore 1996 ). The c-Abl protein tyrosine kinase is activated in the case of double chain DNA breaks (Burger et al 2019 ). It also takes part in regulating cytoskeletal structure, cell division, cell growth and cell proliferation (Hantschel & Superti-Furga 2004 , Plattner et al 1999 ). It has been shown in a study that c-Abl tyrosine kinase directly combines with the telomerase catalytic subunit (hTERT), leading to phosphorylation of tyrosine residues and inhibition of telomerase (Kharbanda et al 2000 ). Alternative pathways used for the treatment of Chronic Myelogenous Leukaemia (CML) have also been shown to link c-Abl and TERT. The fusion protein of Bcr-Abl has been shown to activate Abl tyrosine kinase and potentiate telomerase suppression in Bcr-Abl-positive cells. Also, in the chronic stage of CML, telomerase activity has been shown to depend on drug administration, and in the development of the disease, chemotherapy can accelerate or suppress the loss of telomere length in leukemia cells through direct or indirect (protein kinase-mediated) telomerase control (Bakalova et al 2003 ). Telomeres are special structures located at the chromosome ends and maintain chromosome integrity (Blasco et al 1997 ). Telomerase is formed by the DNA polymerase complex bound to ribonucleoprotein RNA and consists of the RNA template and catalytic protein telomerase reverse transcriptase (TERT) (Hamad et al 2002 ). In our previous study, we identified that c-Abl expression in uterus during mouse estrus cycle, embryonic and placental development in the mouse (Yaba et al 2011 ) We also showed the localizations of c-Abl and mTERT, protein and mRNA levels in prenatal and postnatal development gonadal development (Yildirim & Yaba 2020 ). Additionally, We determined the relationship between c-Abl and mTERT on mouse granulosa cells and we suggested that this interaction is very crucial in mouse folliculogesis (Yaba et al 2020 ). In the purposed study, we aimed to determine the potential role of c-Abl and mTERT in DMBA-induced ovotoxicity. We thought that revealing the possible relationship between c-Abl and mTERT activity would contribute to elucidating the mechanisms underlying rapid oocyte depletion and ovarian senescence in DMBA-induced ovotoxicity in mouse. RESULTS DMBA Induces Ovotoxicity and Causing Development of Atretic Follicles The follicles at different stages of follicular development were determined in the all experimental group ovaries. The DMBA-treated ovaries presented follicles at different stages of follicular development in ovarian cortex (Fig. 1 ). However, the number of developing follicles is higher than primordial and primary follicles that are waiting dormant in early development and the disruptions in the cytoplasm and zona pellucida of oocytes in the follicle occured in DMBA-treated ovary. In a 2017 study with pig oocytes, it was shown that DMBA affects and modifies the meiotic process (Song et al 2017 ). When we examined the nuclei, we observed that there were meiotic errors and some oocytes were not in the prophase I (PI) (Fig. 2 ). We observed that both mitosis and apoptosis are activated in the granulosa cells in the follicles compared to the control group. Also, the atretic follicles seen in the DMBA-induced ovaries were quite striking. (Fig. 2 ). Morphology was first evaluated for ovaries treated with in vitro DMBA. As a result of short-term DMBA-treatment applied in vitro , primordial, growing and atretic follicles were determined. This indicates that follicular development continues (Fig. 3 ). DMBA Increases The Rate of Developing Follicles and Affects the Follicle Reserve After morphological examination, follicle counting were performed in vivo and in vitro ovaries. We determined that the number of primordial and primary follicles decreased dramatically when the DMBA group was compared with the control group in the in vivo DMBA-treated ovary. In addition, there was an increase in the number of follicles in the developing secondary stages compared to the control group. However, a significant increase in the number of atretic follicles was seen in the DMBA-induced ovaries. There was no significant difference in the number of antral and Graafian follicles (p < 0.05) (Fig. 4 A). As a result of in vitro short-term DMBA treatment, there was no difference in the development of primordial follicles. However, a decrease was determined in the number of growing follicles compared to the control group. The number of atretic follicles in the ovaries significantly increased compared to the control group (Fig. 4 B). These results showed that short-term DMBA-treatment significantly affected the number of atretic follicles, in the mouse ovary. Especially after iDMBA treatment, the developing follicles in the ovarian reserve are also affected. Effect of DMBA on AMH Levels in the DMBA-Treated Mouse The Anti-mullerian hormone (AMH) level in serum was measured from the DMBA-treated and control group animals by ELISA. The AMH level increased significantly in the DMBA-treated group. This result confirms that early primordial follicle activation occurs with DMBA-induced ovotoxicity. AMH is secreted by the primary follicle and granulosa cells in the follicles in the early antral follicle stage and is involved in the activation of the primordial follicles (Broekmans et al 2009 ) The decrease in the level of AMH hormone is an indicator of the decrease of the follicle pool in the ovary (Fig. 5 ). DMBA Induce Mithochondrial and Morphological Defects in Mouse Oocyte and Granulosa Cells TEM method was used for ultrastructural evaluation of ovarian tissues isolated from control, DMBA-treated and vehicle groups. As a result, ovarian tissues in the control group and the DMBA-induced group were compared with the following criteria as transzonal projections (TZPs), tubular cristae of mithochondria and apoptosis of granulosa cell. Transzonal projections have very important roles in the communication between oocyte and granulosa cells, in completing maturation of oocyte and in growth of the follicle. In control group, we identified numerous transzonal projections originating from the granulosa cells surface and ending in oocyte surface extending along the zona pellucida (Fig. 6 A). However, the number of transzonal projections decreased and their length was shortened in the DMBA-treated group (Fig. 6 B). Tubular cristae are present in steroid-producing cells. We determined lamellar and tubular associations of the mithochondrial cristae in control group granulosa cells in follicles (Fig. 6 C). The mitochondria of the granulosa cells in the follicles were damaged and they mostly lost their cristae structure in the DMBA-treated group (Fig. 6 D). Additionally, some of the oocytes in the follicles were not at the prophase 1 (PI) stage in DMBA-treated group, and that they reached the metaphase 1 (MI) stage by continuing with meiosis (Fig. 6 E). In addition, there were more apoptotic granulosa cells in the follicles in DMBA-treated group when compared with control group (Fig. 6 F). The findings obtained with TEM show that the oocyte nuclear maturation in DMBA-treated group is disrupted by the ovotoxicity resulting from DMBA exposure and physical communication between oocyte and granulosa cells is impaired and granulosa cells are damaged by the mitochondrial cristae structure. DMBA Increases ROS Production TAS levels were measured in control, vehicle and DMBA groups. The TAS level in the DMBA-treated group was significantly lower than the control group (Fig. 7 A). Considering the TOS levels, the TOS level in the DMBA-treated group was significantly higher compared to control group, unlike the TAS level (Fig. 7 B) (Table 1). Detection of c-Abl and mTERT Localization In the DMBA-Treated Mouse Ovary c-Abl and mTERT protein localization were evaluated with immunofluorescence staining in the control and DMBA-treated mouse ovarian tissue (Fig. 8 A-A’, B-B’, C-C’). We determined that c-Abl and mTERT immunostaining patterns showed the same expression pattern as the control group and DMBA-induced ovotoxicity group. c-Abl and mTERT protein localizations were detected in the granulosa cells and oocyte in Graafian follicle (Fig. 8 A (A-A’, B-B’, C-C’)). In addition, we observed that mTERT immunostaining was more intense in the DMBA-treated group compared to the control group. The mTERT immunoflourescence intensity in both oocyte and granulosa cells was higher than that of c-Abl (Fig. 8 B and C). We also examined the localization of c-Abl and mTERT in in vitro mouse ovaries treated with short-term DMBA. We determined the expression of c-Abl and mTERT in both granulosa cells and oocyte in the developing follicles (Fig. 9 A (A-A’, B-B’, C-C’)) and mTERT immunoflourescence intensity was higher in both granulosa and oocyte compared to c-Abl. In addition, mTERT expression in the DMBA-treated group was compared to the control group, both in the oocyte and in the granulosa cells (Fig. 9 B and C). c-Abl, mTERT, PCNA and Caspase Protein Expression in DMBA-Treated Mouse Ovary We performed western blot experiment to compare protein expression levels between groups. As a result of our western blot experiment with ovarian tissues samples isolated from control, veichle and DMBA treatment groups, c-Abl expression decreased in ovarian tissues isolated from ovotoxicity group by applying in vivo DMBA treatment (**p < 0.005) (Fig. 10 A and B). We determined that mTERT protein expression level increased (**p < 0.005) (Fig. 11 A and B). The PCNA protein level in the DMBA-treated ovary was significantly lower than the control group (***p < 0.0005) (Fig. 12A and B). There was no significant difference in Caspase protein level between experimental groups (Fig. 13A and B). c-Abl and mTERT mRNA Expression In DMBA-Treated Mouse Ovary c-Abl and mTERT mRNA expression levels were measured by qRT-PCR, and all were not changed in control and DMBA-treated groups (Fig. 14A and 14B). DISCUSSION DMBA is an organ-specific enviromntal carcinogen which has a potent negative effects on follicular and ovarian development in mammals (Ganesan & Keating 2014 ), (Ganesan et al 2015 ) and cause ovotoxicity stimulate follicle loss by activating the primordial follicle population. Recent studies have also showed that this ovotoxicity can induce POI through dysfunctional primordial follicle activation (Sobinoff et al 2011 ). In this study, we aimed to evaluate the possible roles of c-Abl and mTERT DMBA-induced ovotoxicity mechanism. The DMBA-treated ovaries presented the decrease in the number of primordial and primary follicles but the increase in the number of secondary follicles shows that DMBA stimulates follicle activation. However, the significantly increased number of atretic follicles in the DMBA-treated group was also remarkably evident. DMBA causes follicular atresia in the entire follicular stages and atretic follicles seen in the DMBA-treated ovaries are quite striking. When we examined the developing follicles in DMBA treated group, we observed deterioration in the cytoplasm and zona pellucida of oocytes compared to the control group. When we examined the nuclei of the oocytes in the atretic follicles of DMBA-induced group, we observed that there were errors in meiotic division. Our findings are consistent with the findings that DMBA exposure causes disruptions in the meiotic process (Song et al 2017 , Yang et al 2020 ). We observed that both mitotic and apoptotic cells were activated in the granulosa cells in the follicles in the ovary treated by DMBA compared to the control group. AMH expression begins in cuboidal granulosa cells of the primary follicles, which are formed after the primordial follicles are recruited from the follicle reserve as dormant follicle, and AMH level increases until the preantral and small antral follicular stages (Durlinger et al 2002 ). In the later follicular stages, AMH expression decreases and is not seen during follicular growth due to follicle stimulationg hormone (FSH). Also, AMH is expressed only in healthy follicles, not in follicles that undergo atresia (Durlinger et al 2002 , Visser et al 2012 ). Decrease in serum AMH levels is associated with the reduction in the size of the antral follicle pool (de Vet et al 2002 , van Rooij et al 2002 ). In this study, we determined that the number of primordial follicles decreased in DMBA-treated mouse ovaries, whereas the number of primary and secondary follicles increased, in parallel, serum AMH levels increased. However, when we evaluated the structure of the developing follicles, we observed that DMBA causes impairment in cytoplasmic and nuclear maturation of oocytes. When we support these findings with the literature, we suggested that DMBA causes premature primordial follicle activation, increasing the number of developing follicles and AMH levels in DMBA-treated group. The TEM findings demonstrated that the number of transzonal projections decreased and their length was shortened in the DMBA-treated group, that the granulosa cells had damage in their mitochondria, and that they mostly lost their crystae structure. In addition, there were too many apoptotic granulosa cells in different stages developing follicles in the DMBA-treated group and errors occurred in the meiotic division of the oocyte, progressing from prophase I (PI) to metaphase I (MI). These findings show that premature follicle activation resulting from DMBA treatment negatively affects both oocyte maturation and the communication between granulosa cells and oocyte, causing impaired follicle development, triggering follicular atresia and leading a decrease in primordial follicle reserve. Based on this, we suggested that DMBA may adversely affect oocyte maturation and quality by inducing disruption of follicle development and meiotic division. Antioxidant enzymes play an important role in defending free radical-mediated tissue or cellular damage (Nampoothiri et al 2007 ). TAS level in ovotoxicity induced by DMBA was found to be lower than the control group. However, TOS level was found to be higher in the DMBA-treated group compared to the control group. As a result, we suggested that DMBA increases the oxidative stress level in the ovary and blocks antioxidant systems. Telomerase activity and elongation of telomeres are necessary for proliferation of granulosa cells, especially in small and growing follicles (Liu & Li 2010 ). In a study, it was determined that estrogen positively regulates TERT gene expression by acting directly on the TERT gene promoter region and indirectly through the MYC gene (Liu & Li 2010 ). In addition, it has been shown that estrogen receptor 1 and 2 are involved in the regulation of the transcription of the TERT gene in cancer and healthy differentiated cells (Grasselli et al 2008 , Kondoh et al 2007 ). In our study, we determined an increase in the number of follicles in the secondary stage and a decrease in mTERT expression as a result of premature follicle activation in DMBA-induced ovotoxicity. Studies have shown that telomerase is short and telomerase activity is low in leukocytes and granulosa cells isolated from peripheral blood in women with POI (Butts et al 2009 , Xu et al 2017 ). In addition, telomeric shortening of granulosa cells and decreased telomerase activity in young women have been shown to cause ovarian failure (Butts et al 2009 ). This literature information confirms our datas. However, considering telomerase, germ cell growth and neoplastic immortalization, it is like a double-edged knife. It will be useful to observe how the proliferating granulosa cells will undergo a change with the effect of DMBA in the future with a longer exposure to DMBA. We determined that DMBA decreased its expression by inhibiting c-Abl in ovarian tissue treated with DMBA for one week. The fact that DMBA causes premature follicle activation and as a result of this, the increase in the number of follicles in the secondary stage shows us that oocytes and granulosa cells get rid of the repair mechanism due to DMBA suppressing c-Abl and they can continue their development by choosing an alternative pathway. The fact that there was no significant change in Caspase3 expression between the groups supports this finding. A study has shown that the carcinogenic effect of DMBA can be treated by applying the c-Abl inhibitor Imatinib (Morgan et al. 2013). In the light of the findings, we suggested that oocyte and granulosa cells escape from the DNA repair control mechanism by suppressing c-Abl after DMBA treatment and thus premature activated follicles develop into the secondary stage as the granulosa cells continue their proliferation. In our results; we showed that ovotoxicity caused by DMBA-treatment may cause premature depletion of developing follicles due to follicle activation. An increase in developing follicles is an increase in the total number of developed follicles. The increasing number of atretic follicles after DMBA-treatment presented disruption of communication between oocyte and granulosa cells, and damage to the nucleus during meiotic division. We showed that DMBA has an inhibitory effect on c-Abl expression in mouse ovary after 1 week of treatment, while it has a triggering effect on mTERT. We determined that c-Abl and mTERT have a potential role in DMBA-induced ovotoxicity. However, when the functional role of c-Abl and mTERT in ovotoxicity is investigated, we will have more detailed information about the molecular mechanisms. MATERIAL AND METHODS All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YÜDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. All experiments were performed in accordance with guidelines and regulations of Faculty of Medicine of Yeditepe University. Generation of DMBA-treated mouse model In vivo DMBA treatment Female BalbC mice (n = 10 for each group) at 28 day of age, were obtained from the YUDETAM. A total of 4–5 mice per cage were maintained for 14 days in a temperature-controlled environment under 12h:12h light-dark cycles with ad libitum access to food and water. To induce ovotoxicity, mice were administered daily intraperitoneal injection (ip) of consecutive doses of either sesame oil containing vehicle control (< 10 µl/kg/daily sesame oil) or sesame oil containing DMBA 7,12-dimethylbenz(a)anthracene (DMBA) (Lgc standards; #DRE-C20745000) (1mg/kg/Daily dissolved in sesame oil) for 14 days (Borman et al 2000 ). Mice were disected after 1 week from last treatment day. Right ovaries from each experimental group were used morphological evaluation (Hematoxylin and Eosin (H&E) staining and follicle counting (n = 10), immunofluorescence staining and ultrastructural investigation) and left ovaries were used for western blot (n = 5) and qRT-PCR experiment (n = 5). In vitro DMBA treatment To determine the effect of DMBA in vitro , the ovaries were dissected at 28 day and cultured in 6-well culture plates (n = 6 for each group). Ovaries cultured with RPMI medium containing 10% FBS, 7,5 mg Ascorbic Acid, 1,25 ml L-Glutamine, 75 µl, 250 µl FGF (fibroblast growth factor), 30 µl LIF (Leukemia inhibitory factor), 37,7 µl SCF (stem cell factor), 3 ml B27 and penicillin/streptomycin for 4 days at 37°C and 5% CO 2 , culture media changes every 2 days. Ovaries were treated with control medium (no application), vehicle medium (0.01% acetone) and DMBA (50 nM dissolved in acetone) (Sobinoff et al 2011 ). Tissue Processing and Hematoxylin and Eosin Staining All ovarian tissue samples were fixed with 10% neutral buffered formalin for 18 hours, dehydrated using a graded series of 70, 80, 90 and 100% alcohol, vitrified in xylene and embedded in 60°C paraffin. 5µm serial sections were taken by microtome (Leica, #RM2245) onto poly-L-lysine coated slides (Thermo Scientific, #P4981). Paraffin sections were deparaffinized in xylene, rehydrated in alcohol series (100, 90, 80 and 70%). Sections were stained with H&E staining (Bio-optica Gill's hematoxylin; #06014/L, Bio-optica Eosin Y alcoholic solution; #10003/L). Then passed through the increased alcohol series, covered with mounting medium and examined under light microscope (Leica, #DM6000) (Yu et al 2011 ). Follicle counting The number of follicles in all the serial sections of an in vivo and in vitro ovaries were counted (Greenfeld et al 2007 ). H&E staining was performed in serial sections to evaluate ovarian morphology and determine the number of primordial, primary, secondary, preantral and atretic follicles in the ovaries. 5 µm thickness sections were taken from paraffin blocks and the only follicles with visible nucleus seen in the oocyte were counted in each section. Follicles are different follicular stages were determined by counting from each group. After the total number of follicles was determined, the results of follicle counts between the groups were compared. For in vivo groups; primordial follicle was based on having an oocyte surrounded by a single layer of flat pregranulosa layer. Primary follicles were defined as an oocyte surrounded by a single layer of cuboidal granulosa cells. Secondary follicles were identified by presence of two or three layers of cuboidal granulosa cells surrounding the oocyte. It was defined by the emergence of small follicular spaces between granulosa cells in preantral follicles. Atretic follicle determined by a follicle that enters a degenerative process without ovulation (Greenfeld et al 2007 ). For in vitro groups; follicles with a clearly visible nucleus were counted. Primordial, growing and atretic follicles were determined for the in vitro group. These follicles were classified as primordial follicles (flat pre-granulosa cells surrounding the oocyte), growing follicles (an enlarged oocyte surrounded by multiple cuboidal granulosa cells), and atretic follicles (a degenerate oocyte with collapse cytoplasm) (Zhang et al 2022 ). Detection of Anti-Müllerian Hormone (AMH) levels in DMBA-Treated mouse model serum. After dissection, blood samples were obtained by cardiac puncture from mice all groups to measure the Anti-Müllerian Hormone (AMH) levels. The blood samples were centrifuged at 4000 rpm for 15 minutes. The serum was isolated, frozen and kept at − 80°C until analysis. AMH level was measured by ELISA kit (Mouse AMH ELISA Kit, Sunred, #DZE201021297) according to the manufacturer’s instructions. Briefly, serum samples were centrifuged and then standards were prepared. The absorbance of each well was measured Microplate Spectrophotometer 450 nm wave length using a microplate reader through the Gen5 program. Transmission Electron Microscopy Transmission electron microscopy (TEM) was used to examine oocyte and follicle cells at the ultrastructural level ovarian tissues isolated from control (n = 3), vehicle (n = 3) and DMBA-treated group (n = 3). Ovarian tissues were trimmed approximately 1mm 3 in size and fixed in 4% Gluteraldehyde (prepared in 0.1 M Sorensen’s Phosphate Buffer (SBP)) for 2 hours. Then, it was washed with SBP and fixed with osmium tetraoxide (OsO 4 ) fixative prepared in 1% SPB. The tissues were then dehydrated. Subsequently, after incubating for 1 hour in 1% uranyl acetate solution prepared with 75% ethanol, 2 times for 10 minutes in Propylene oxide, 2 times in Propylene oxide + (Araldite master mix + 2% accelerator BDMA) (1:1) mixture. Incubated at room temperature for 4 hours. Tissues that were incubated overnight in araldit were buried fresh araldite the next day and kept at 60 o C for 48 hours. Sections taken from araldit blocks using ultramicrotome were visualized under a TEM microscope (ZEİSS-LEO 906E). Determination of Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) groups were performed using Rel Assay Diagnostic Kits. For TAS analysis, 30 seconds were waited and then measured with 660 nm wavelength. Reagent2 was then added and incubated for 5 min and measured with 660 nm wavelength. For TOS analysis, it was waited 30 seconds after adding reagent1 to serum samples and then measured with 530 nm wavelength. Then reagent2 was added and it was incubated for 5 min and measured with 530 nm wavelength. Measurements were made with Bio-tek SynergyTM HT Multi-Detection Microplate Reader device. Immunofluorescence staining 5 µm ovarian tissue sections were incubated in a 37°C incubator for 1h. Tissues were permeabilized and rehydrated with descending alcohol series and tissue sections were encircled with a histology pap pen to maintain staining solutions concentrated on the tissue during processing. Tissues were permeabilized in TBS with 0.1% Tween 20 (TBST), followed by antigen unmasking (Citrate Buffer) and blocking (TBS-T, NGS/5%) for 60 min at room temperature. After blocking tissues were incubated with c-Abl (PA5, 39688, Rabbit, Thermo, 1:100) primary antibody for 2 hours at room temperature. After washing with TBS-T, secondary antibody (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed, Abcam, ab96883, 1:250) was placed for 1.5 hours at room temperature. Afterwards for double immunoflourescence staining, sections were blocked in and incubated mTERT (MA5-16034, Mouse mA6, Thermo, 1:100) primary antibody overnight at 4°C. The next day the sections were washed with TBS-T for 3 times for 5 minutes and incubated with secondary antibody (Goat Anti-Mouse IgG H&L Alexa Fluor® 647, Abcam (ab150115, 1:250) for 1,5 hour at room temperature. Sections were mounted with a DAPI mounting medium and visualized with Confocal microscope (Zeiss, LSM780) (Yaba et al 2020 ). Western Blot Analysis After the samples were homogenized, protein was measured from tissue lysates. The total proteins were subjected to electrophoresis in %4–12 Bis-Tris gels (ThermoFisher Scientific; NP0321) and transferred onto a PVDF membrane (ThermoFisher Scientific; IB401001) using the overnight wet transfer system. Subsequently, membranes were incubated overnight at 4°C with c-Abl (Sigma #A5844, 1:1000), mTERT (Santa Cruz #sc-7212, 1:1000), Caspase3 (D3R6Y, #14214S Cell signaling, 1:1000) and PCNA (D3H8P, #13110S Cell signaling, 1:1000) and Beta-actin (Cell Signaling #8H10D10, 15000) polyclonal antibodies. After incubation with primary antibody, membranes were washed 3 times for 5 minutes with TBS-T. Incubated with secondary antibody for 2 hours at room temperature and membranes viewed with CCD camera (Yaba et al 2020 ) Quantitative RT-PCR The total RNA was extracted from the in vivo and in vitro DMBA-treated ovarian tissues by using a Total RNA Purification Kit (Jena Bioscience) according to manufacturer’s procedure. The concentration and purity of the isolated total RNA was determined spectrophotometer (Bio-tek SynergyTM HT Multi-Detection Microplate Reader). Total RNA was reverse transcribed to cDNA with an SCRIPT cDNA Synthesis Kit (Jena Bioscience) according to manufacturer’s protocol with an OligodT primer. The target fragments were quantified by real-time PCR with a qPCR GreenMaster with UNG (Jena Bioscience) with 2µl of the cDNA template in a Real-Time PCR Detection System (Bio Rad). qRT-PCR was carried out using the following parameters: 5 min initial denaturation at 94 o C, followed by 30 cycles (amplification) at 92 o C for 20 s, 59 o C for 20 s, and 72 o C for 1 min. Melt curve analyses were run with each series to confirm the specificity of the amplified products. The standard curves were prepared for c-Abl (F 5’-CGG GAC CAT GTT GGA GAT-3’, R 5’-TTC ATA CCG CAG CGA GATG-3’), mTERT (F 5'-CCTGCGGCCCATTGTGAAC-3', R 5'-GTG GAC TTG GCC TTG GCT ATC TCT-3') and Beta-actin (F 5’-GACCTCTATG- CAACACAGT-3’, R 5’ -TTG CTG ATC CAC ATC TGCT-3’). Each sample was tested in duplicate. The gene expression datas were normalized to β-actin expression (Yaba et al 2020 ). Significant differences determined by two-way ANOVA test. Statistical analysis For immunofluorescence intensity of c-Abl and mTERT expression and western blot results were analyzed by using ImageJ and statistical analysis was performed with GraphPad Prism. For the immunofluorescence intensity results data were analysed by Unpaired t test with Welch’s correction. For the WB results were analysed by a One-Way ANOVA test. For follicle counting results datas were analysed by Two-Way ANOVA test to compare multiple groups. For the qRT-PCR results were analysed by a Two-Way ANOVA test (*p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0005). Abbreviations POI: Premature Ovarian Infufficiency DMBA: 7,12-Dimethylbenz[a]anthracene c-Abl : Abelson Tyrosine Kinase TERT: Telomerase Catalytic Subunit H&E: Hematoxylin and Eosin AMH: Anti-Müllerian Hormone TEM: Transmission electron microscopy SBP: Sorensen’s Phosphate Buffer TAS: Total Antioxidant Status TOS: Total Oxidant Status P1: Prophase 1 MI: Metaphase 1 TZPs : Transzonal Projections Declarations Ethics approval All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. All procedures carried out in this study were conducted according to the Yeditepe University Experimental Animals Ethics Directive which has been prepared according to the rules and principles in Universal Declaration of Animal Rights, Competing Interests The authors declare no competing interests. Corresponding author Correspondence to Aylin Yaba. Funding statement This project supported by from The Scientific and Technological Research Council of Turkey (TÜBİTAK#215S867). Author Contribution E.Y performed western blot, ELISA, TAS/TOS. T.O. performed H&E staining, follicle counting and qRT-PCR. A.Y. performed TEM analysis, conceived and coordinated the study. A.Y, E.Y. and T.O. performed analysis of all experimental results and wrote the paper. Acknowledgement This project supported by from The Scientific and Technological Research Council of Turkey (TÜBİTAK#215S867). Data Availability All relevant data are within the paper. References Bakalova R, Ohba H, Zhelev Z, Ishikawa M, Shinohara Y, Baba Y (2003) Cross-talk between Bcr-Abl tyrosine kinase, protein kinase C and telomerase-a potential reason for resistance to Glivec in chronic myelogenous leukaemia. 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Hum Reprod 32:201–207 Yaba A, Agus S, Yildirim E, Erdogan CS, Yilmaz B (2020) Interaction of the mTERT telomerase catalytic subunit with the c-Abl tyrosine kinase in mouse granulosa cells. Journal of receptor and signal transduction research 40: 365 – 73 Yaba A, Kayisli UA, Johnson J, Demir R, Demir N (2011) The Abelson tyrosine kinase (c-Abl) expression on the mouse uterus and placenta during gestational period. J Mol Histol 42:91–96 Yang CX, Song ZQ, Pei S, Yu XX, Miao JK et al (2020) Single cell RNA-seq reveals molecular pathways altered by 7, 12-dimethylbenz[a]anthracene treatment on pig oocytes. Theriogenology 157:449–457 Yildirim E, Yaba A (2020) Determination of c-Abl tyrosine kinase and mTERT catalytic subunit of telomerase expression level during prenatal-postnatal mouse ovary-testis development. Reprod Biol 20:555–567 Yu J, Yaba A, Kasiman C, Thomson T, Johnson J (2011) mTOR controls ovarian follicle growth by regulating granulosa cell proliferation. PLoS ONE 6:e21415 Zhang X, Zhang W, Wang Z, Zheng N, Yuan F et al (2022) Enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling. Cell Death Dis 13:87 Zhu J, Shore SK (1996) c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein. Mol Cell Biol 16:7054–7062 Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.tiff Table 1: Follicle counting results. Table2.tiff Table 2: Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) levels in DMBA-treated, control and vehicle groups. Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2025 Read the published version in Journal of Molecular Histology → Version 1 posted Editorial decision: Revision requested 21 Oct, 2025 Reviews received at journal 16 Oct, 2025 Reviews received at journal 28 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 14 Sep, 2025 Editor assigned by journal 09 Sep, 2025 Submission checks completed at journal 09 Sep, 2025 First submitted to journal 09 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7571888","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":518160419,"identity":"60a984e9-f069-4c63-9e83-57bb5fa4449b","order_by":0,"name":"Ecem Yildirim","email":"","orcid":"","institution":"Yeditepe University","correspondingAuthor":false,"prefix":"","firstName":"Ecem","middleName":"","lastName":"Yildirim","suffix":""},{"id":518160420,"identity":"95b3dc58-4af5-4bc8-afed-2721029a473c","order_by":1,"name":"Tugce Onel","email":"","orcid":"","institution":"Yeditepe 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09:00:50","extension":"html","order_by":63,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127421,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/7fa1bc36779a870c3acbdd39.html"},{"id":91830168,"identity":"433f2e65-6ba1-4cb6-b450-6fab268416f6","added_by":"auto","created_at":"2025-09-22 09:00:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21888954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological evaluation of primordial, primary, secondary and antral follicle for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003econtrol and DMBA-treated group. \u003c/strong\u003eMorphological evaluation of in vivo control ovaries and DMBA-treated ovaries performed with Hematoxylin\u0026amp;Eosin staining. Follicles in primordial, primary, secondary and antral stages were shown for all groups.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/d1ac6b90c080721a60cf3b9f.png"},{"id":91828105,"identity":"357e412d-b5e6-4cd6-833c-b9f33ad05394","added_by":"auto","created_at":"2025-09-22 08:52:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8729111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological evaluation of atretic follicles in vivo DMBA-treated group. \u003c/strong\u003eMorphological evaluation DMBA treated ovaries performed with Hematoxylin\u0026amp;Eosin staining. Atretic follicles were shown for all groups. \u003cstrong\u003e(A)\u003c/strong\u003e a follicle with disrupted oocyte membrane structure \u003cstrong\u003e(B)\u003c/strong\u003e is a secondary follicle where disruptions occur in the oocyte nucleus (black arrow). \u003cstrong\u003e(C)\u003c/strong\u003e vacuolization within the oocyte \u003cstrong\u003e(D)\u003c/strong\u003e an atretic follicle that cannot complete its development is observed DMBA treated ovary (40X).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/d21a34f23e80d4b1f4e1a09f.png"},{"id":91830170,"identity":"227517be-3176-40cf-91ad-1f3801445c92","added_by":"auto","created_at":"2025-09-22 09:00:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9553405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological evaluation of primordial, primary, secondary and antral follicle for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e control and DMBA-treated group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphological evaluation of \u003cem\u003ein vitro\u003c/em\u003e control ovaries and DMBA-treated ovaries performed with Hematoxylin\u0026amp;Eosin staining. Follicles in primordial, growing and antral stages were shown for all groups.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/5c8c2b8ff32e0da505b325ab.png"},{"id":91830144,"identity":"28c209f3-9369-4467-b5c4-06c15beab85e","added_by":"auto","created_at":"2025-09-22 09:00:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFollicle counting. A. \u003c/strong\u003eComparison of the numbers of primordial, primary, secondary, antral, graaf and atretic follicles in the \u003cem\u003ein vivo\u003c/em\u003e experimental groups. In the DMBA, vehicle and control group ovary, follicle count was performed based on the organization of the follicles surrounding the oocyte. Considering the results, the number of primordial follicles decreased significantly in the DMBA group compared to the control group (***p \u0026lt;0.0005). The number of primary follicles decreased significantly compared to the DMBA group (***p \u0026lt;0.0005). The number of secondary follicles increased significantly compared to the DMBA group (* p\u0026lt;0.05). \u003cstrong\u003eB.\u003c/strong\u003eComparison of the numbers of primordial, growing and atretic follicles in the \u003cem\u003ein vitro\u003c/em\u003e experimental groups. Considering the results, the number of growing follicles decreased significantly in the \u003cem\u003ein vitro\u003c/em\u003e DMBA group compared to the control group (***p \u0026lt;0.0005). The number of primary follicles decreased significantly compared to the DMBA group (***p \u0026lt;0.0005). The number of secondary follicles increased significantly compared to the DMBA group (* p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/50090281a9ff8dbfb649b8d4.png"},{"id":91830145,"identity":"2c29ffe8-9644-4c29-b63a-0df405569de2","added_by":"auto","created_at":"2025-09-22 09:00:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum Anti-Müllerian Hormone (AMH) levels were evaluated with using ELISA. \u003c/strong\u003eThe animals were divided into control, vehicle and experimental groups as follows: i) An untreated Control group (wild-type; WT) of n=10 mice; ii) Vehicle group of n=10 DMBA-treated mice receiving a sesame oil injection 100 µl; iii) DMBA experimental group of 1mg/kg DMBA-treated mice, injected daily with 100 µl of either. After treatment with DMBA, Anti-Müllerian hormone levels increased significantly compared to the vehicle and control group (*p\u0026lt;0.05). There was no significant difference between the other groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/aed889472d69642fff825f07.png"},{"id":91830161,"identity":"3517e772-84b6-4927-9fcc-a9ba70cd1f58","added_by":"auto","created_at":"2025-09-22 09:00:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":32365611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranszonal extensions (white arrow) in control group\u003c/strong\u003e (A). Transzonal extensions with decreased and shorter length were observed in the DMBA-induced ovotoxicity (B). Numerous mitochondria observed in normal morphology with tubular crystals in granulosa cells in ovary tissues taken from the control group (C). Damage to the mitochondria of granulosa cells in the DMBA-treated group (D). gc: Granulosa cell, area delimited by blue line: tubular crystalline mitochondria, blue arrows: damaged mitochondria. An oocyte at metaphase 1 (MI) in the DMBA-treated group (E). An apoptotic granulosa cell surrounding the oocyte (F). White dashed line: chromatins, (*): apoptotic granulosa cell, o: oocyte, zp: zona pellucida.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/0783bcd8d1708376fb7a7ba6.png"},{"id":91828069,"identity":"a3fcc870-5c9a-45b4-8abb-5206ab758c42","added_by":"auto","created_at":"2025-09-22 08:52:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":528405,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal Antioxidant Status (TAS) and Total Oxidant Status (TOS) levels were evaluation by test kits.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal Antioxidant Status (TAS) \u003cstrong\u003e(A)\u003c/strong\u003e and Total Oxidant Status (TOS) \u003cstrong\u003e(B)\u003c/strong\u003e levels in DMBA-treated ovary induced by DMBA treatment. DMBA treated groups TAS levels decreased significantly compared to the control group (A) (**p\u0026lt;0.005). While the TOS levels of DMBA treated groups increased significantly compared to the control group (B) (***p \u0026lt;0.0005).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/981d6d73736c1c012eb97bfa.png"},{"id":91830162,"identity":"51a28323-1803-4ea5-960d-befb2f5a87f6","added_by":"auto","created_at":"2025-09-22 09:00:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":8410750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and localization of c-Abl and mTERT in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e experimantal ovarian tissue.\u003c/strong\u003e \u0026nbsp;\u003cstrong\u003eA.\u003c/strong\u003e Immunoflourescence staining and confocal microscopy showing expression and localization of c-Abl \u003cstrong\u003e(A, A’)\u003c/strong\u003e and mTERT \u003cstrong\u003e(B, B’)\u003c/strong\u003e for in vivo ovarian tissues using series sections of ovary tissues from DMBA-treated and control group. Nucleus imaging was performed with DAPI staining\u003cstrong\u003e. \u003c/strong\u003ec-Abl / mTERT / DAPI overlapped image is displayed \u003cstrong\u003e(C, C’). \u003c/strong\u003e(Scale Bars= 50μm)\u003cstrong\u003e.\u003c/strong\u003e Quantitative analysis of c-Abl and mTERT densities in DMBA and control granulosa cells \u003cstrong\u003e(B)\u003c/strong\u003e and oocyte \u003cstrong\u003e(C).\u003c/strong\u003e There was no significant difference c-Abl and mTERT expression levels of granulosa cells between the groups \u003cstrong\u003e(B)\u003c/strong\u003e. The expression level of mTERT in the DMBA group was significantly higher compared to the control group. In addition, mTERT expression level was significantly higher than c-Abl expression level in both groups \u003cstrong\u003e(C) \u003c/strong\u003e(***p \u0026lt;0.0005).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/8aec882980f6cf16ccbc303b.png"},{"id":91828124,"identity":"7a7a278b-a96e-498d-96a5-fcef11b27236","added_by":"auto","created_at":"2025-09-22 08:52:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":9190909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression and localization of c-Abl and mTERT in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e experimantal ovarian tissue. A. \u003c/strong\u003eImmunoflourescence staining and confocal microscopy showing expression and localization of c-Abl \u003cstrong\u003e(A, A’)\u003c/strong\u003e and mTERT \u003cstrong\u003e(B, B’)\u003c/strong\u003e for \u003cem\u003ein vitro\u003c/em\u003e ovarian tissues from DMBA-treated and control group. Nucleus imaging was performed with DAPI staining\u003cstrong\u003e. \u003c/strong\u003ec-Abl / mTERT / DAPI overlapped image is displayed \u003cstrong\u003e(C, C’).\u003c/strong\u003e (Scale Bars= 20μm, Scale Bars= 50μm)\u003cstrong\u003e. \u003c/strong\u003eThere was no significant difference c-Abl and mTERT expression levels of granulosa cells between the groups \u003cstrong\u003e(B)\u003c/strong\u003e. There was no significant difference c-Abl and mTERT expression levels of granulosa cells between the groups. The expression level of mTERT in the DMBA group was significantly higher compared to the control group in oocyte. In addition, mTERT expression level was significantly higher than c-Abl expression level in both groups \u003cstrong\u003e(C) \u003c/strong\u003e(***p \u0026lt;0.0005).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/9e6f8051fdfbbec81b313abc.png"},{"id":91828063,"identity":"f3629287-13ff-43f2-ba8f-2ac0536892e9","added_by":"auto","created_at":"2025-09-22 08:52:49","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3574931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ec-Abl, mTERT, PCNA and Caspase-3 protein expression levels in vivo groups were evaluation by western blot\u003c/strong\u003e. \u003cstrong\u003e(A, C, E, and G) \u003c/strong\u003eRepresentative immunoblot image for c-Abl, mTERT, PCNA and Caspase-3. The result of statistical analyzes of protein levels for c-Abl \u003cstrong\u003e(B)\u003c/strong\u003e, mTERT \u003cstrong\u003e(D)\u003c/strong\u003e, PCNA \u003cstrong\u003e(F)\u003c/strong\u003e and Caspase-3 \u003cstrong\u003e(H).\u003c/strong\u003eBeta actin was used as control for protein loading. (**p \u0026lt;0.005, ***p \u0026lt;0.0005)\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/544f09d9a58ed30d196bc6e7.png"},{"id":91830147,"identity":"c06aab9f-5803-4567-8c51-516dae2ae2b4","added_by":"auto","created_at":"2025-09-22 09:00:49","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2417621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ec-Abl and mRNA levels were evaluated with using qRT-PCR \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e groups. \u003c/strong\u003eThere was no significant difference between the DMBA, vehicle and control groups for \u003cem\u003ec-Abl \u003c/em\u003e(A) and \u003cem\u003emTERT \u003c/em\u003e(B\u003cem\u003e)\u003c/em\u003e mRNA levels. Beta actin was used as internal control. Similar results were obtained in three additional experiments.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/699d214e0b8d20c067379892.png"},{"id":97179412,"identity":"0c9b702c-f240-42a7-8aab-3922db137bef","added_by":"auto","created_at":"2025-12-01 16:15:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":103258206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/461044f0-5028-4629-b0b9-2d7dfcd5a822.pdf"},{"id":91828081,"identity":"a954f96d-e0e6-426c-be6b-60a3eefc5d50","added_by":"auto","created_at":"2025-09-22 08:52:49","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3054202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: Follicle counting results.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/bba7467db09dd81ac7737d89.tiff"},{"id":91828094,"identity":"d1a03363-6c2a-4f41-959f-864eb2e9af90","added_by":"auto","created_at":"2025-09-22 08:52:49","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1409822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2: Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) levels in DMBA-treated, control and vehicle groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7571888/v1/571fc566f1f2941a97d351aa.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detection of c-Abl and mTERT Expression by DMBA-Induced Mouse Model of Premature Ovarian Insufficiency","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe oocyte reserve that occurs in prenatal life in mammals decreases and depletes during postnatal life. During this depletion period, a very small part of the oocytes complete their functional development and ovulate, while thousands are eliminated before ovulation. With this mechanism called follicular atresia, ovarian aging occurs and the primordial follicle pool is depleted (Borman et al \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Broekmans et al \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Hansen et al \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Hoyer \u0026amp; Sipes \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and this depletion results infertility or Premature Ovarian Infufficiency (POI).\u003c/p\u003e\u003cp\u003ePolycyclic aromatic hydrocarbon 7,12-Dimethylbenz[a]anthracene (DMBA) is an environmental carcinogen that triggers many tumors, is a widely studied model carcinogen for the leading to a decrease in ovarian reserve and premature ovarian failure in rodents (Hoyer et al \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). So how can people be exposed to DMBA in daily life? As the source of DMBA, it is stated that smoking, consuming foods exposed to activated carbon while cooking (such as barbecued food) and inhaling exhaust fumes from automobiles (Gelboin \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). In addition to its carcinogenic effect, it is also stated that DMBA disrupts the normal process of folliculogenesis, causing a decrease in the follicle population and causing POI (Mattison \u0026amp; Nightingale \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Studies have shown that DMBA causes the activation of the proapoptotic Bax protein and Caspase-3 protein in preovulatory follicles, resulting in the apoptosis of granulosa and theca cells (Tsai-Turton et al \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). When smokers and non-smokers were compared, it was determined that early menopause was induced in smokers (Harlow \u0026amp; Signorello \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Jick \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Mattison et al \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). In addition, it has been shown that the offspring of smoking mothers, even if they do not smoke during pregnancy, have problems such as a decrease in oocyte number, abnormal ovarian function, decreased fertility and/or premature menopause (Jurisicova et al \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In a study with pig oocytes, it was shown that; DMBA triggers changes of meiotic cycle, double strand break, early apoptosis of cumulus cells and histone methylation. At the same time, DMBA causes a significant increase in reactive oxygen species in oocytes both in the presence and absence of cumulus cells around the oocytes (Song et al \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAbelson Tyrosine Kinase (c-Abl) proto-oncogene is a member of a non-receptor protein tyrosine kinase family that provides its biological effects through kinase activity (Zhu \u0026amp; Shore \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The c-Abl protein tyrosine kinase is activated in the case of double chain DNA breaks (Burger et al \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It also takes part in regulating cytoskeletal structure, cell division, cell growth and cell proliferation (Hantschel \u0026amp; Superti-Furga \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Plattner et al \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). It has been shown in a study that c-Abl tyrosine kinase directly combines with the telomerase catalytic subunit (hTERT), leading to phosphorylation of tyrosine residues and inhibition of telomerase (Kharbanda et al \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Alternative pathways used for the treatment of Chronic Myelogenous Leukaemia (CML) have also been shown to link c-Abl and TERT. The fusion protein of Bcr-Abl has been shown to activate Abl tyrosine kinase and potentiate telomerase suppression in Bcr-Abl-positive cells. Also, in the chronic stage of CML, telomerase activity has been shown to depend on drug administration, and in the development of the disease, chemotherapy can accelerate or suppress the loss of telomere length in leukemia cells through direct or indirect (protein kinase-mediated) telomerase control (Bakalova et al \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTelomeres are special structures located at the chromosome ends and maintain chromosome integrity (Blasco et al \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Telomerase is formed by the DNA polymerase complex bound to ribonucleoprotein RNA and consists of the RNA template and catalytic protein telomerase reverse transcriptase (TERT) (Hamad et al \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In our previous study, we identified that c-Abl expression in uterus during mouse estrus cycle, embryonic and placental development in the mouse (Yaba et al \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) We also showed the localizations of c-Abl and mTERT, protein and mRNA levels in prenatal and postnatal development gonadal development (Yildirim \u0026amp; Yaba \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, We determined the relationship between c-Abl and mTERT on mouse granulosa cells and we suggested that this interaction is very crucial in mouse folliculogesis (Yaba et al \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the purposed study, we aimed to determine the potential role of c-Abl and mTERT in DMBA-induced ovotoxicity. We thought that revealing the possible relationship between c-Abl and mTERT activity would contribute to elucidating the mechanisms underlying rapid oocyte depletion and ovarian senescence in DMBA-induced ovotoxicity in mouse.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDMBA Induces Ovotoxicity and Causing Development of Atretic Follicles\u003c/h2\u003e\u003cp\u003eThe follicles at different stages of follicular development were determined in the all experimental group ovaries. The DMBA-treated ovaries presented follicles at different stages of follicular development in ovarian cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the number of developing follicles is higher than primordial and primary follicles that are waiting dormant in early development and the disruptions in the cytoplasm and zona pellucida of oocytes in the follicle occured in DMBA-treated ovary. In a 2017 study with pig oocytes, it was shown that DMBA affects and modifies the meiotic process (Song et al \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). When we examined the nuclei, we observed that there were meiotic errors and some oocytes were not in the prophase I (PI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We observed that both mitosis and apoptosis are activated in the granulosa cells in the follicles compared to the control group. Also, the atretic follicles seen in the DMBA-induced ovaries were quite striking. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMorphology was first evaluated for ovaries treated with \u003cem\u003ein vitro\u003c/em\u003e DMBA. As a result of short-term DMBA-treatment applied \u003cem\u003ein vitro\u003c/em\u003e, primordial, growing and atretic follicles were determined. This indicates that follicular development continues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDMBA Increases The Rate of Developing Follicles and Affects the Follicle Reserve\u003c/h3\u003e\n\u003cp\u003eAfter morphological examination, follicle counting were performed \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e ovaries. We determined that the number of primordial and primary follicles decreased dramatically when the DMBA group was compared with the control group in the \u003cem\u003ein vivo\u003c/em\u003e DMBA-treated ovary. In addition, there was an increase in the number of follicles in the developing secondary stages compared to the control group. However, a significant increase in the number of atretic follicles was seen in the DMBA-induced ovaries. There was no significant difference in the number of antral and Graafian follicles (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs a result of \u003cem\u003ein vitro\u003c/em\u003e short-term DMBA treatment, there was no difference in the development of primordial follicles. However, a decrease was determined in the number of growing follicles compared to the control group. The number of atretic follicles in the ovaries significantly increased compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results showed that short-term DMBA-treatment significantly affected the number of atretic follicles, in the mouse ovary. Especially after iDMBA treatment, the developing follicles in the ovarian reserve are also affected.\u003c/p\u003e\n\u003ch3\u003eEffect of DMBA on AMH Levels in the DMBA-Treated Mouse\u003c/h3\u003e\n\u003cp\u003eThe Anti-mullerian hormone (AMH) level in serum was measured from the DMBA-treated and control group animals by ELISA. The AMH level increased significantly in the DMBA-treated group. This result confirms that early primordial follicle activation occurs with DMBA-induced ovotoxicity. AMH is secreted by the primary follicle and granulosa cells in the follicles in the early antral follicle stage and is involved in the activation of the primordial follicles (Broekmans et al \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) The decrease in the level of AMH hormone is an indicator of the decrease of the follicle pool in the ovary (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDMBA Induce Mithochondrial and Morphological Defects in Mouse Oocyte and Granulosa Cells\u003c/h3\u003e\n\u003cp\u003eTEM method was used for ultrastructural evaluation of ovarian tissues isolated from control, DMBA-treated and vehicle groups. As a result, ovarian tissues in the control group and the DMBA-induced group were compared with the following criteria as transzonal projections (TZPs), tubular cristae of mithochondria and apoptosis of granulosa cell.\u003c/p\u003e\u003cp\u003eTranszonal projections have very important roles in the communication between oocyte and granulosa cells, in completing maturation of oocyte and in growth of the follicle. In control group, we identified numerous transzonal projections originating from the granulosa cells surface and ending in oocyte surface extending along the zona pellucida (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, the number of transzonal projections decreased and their length was shortened in the DMBA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Tubular cristae are present in steroid-producing cells. We determined lamellar and tubular associations of the mithochondrial cristae in control group granulosa cells in follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The mitochondria of the granulosa cells in the follicles were damaged and they mostly lost their cristae structure in the DMBA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Additionally, some of the oocytes in the follicles were not at the prophase 1 (PI) stage in DMBA-treated group, and that they reached the metaphase 1 (MI) stage by continuing with meiosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In addition, there were more apoptotic granulosa cells in the follicles in DMBA-treated group when compared with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). The findings obtained with TEM show that the oocyte nuclear maturation in DMBA-treated group is disrupted by the ovotoxicity resulting from DMBA exposure and physical communication between oocyte and granulosa cells is impaired and granulosa cells are damaged by the mitochondrial cristae structure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDMBA Increases ROS Production\u003c/h3\u003e\n\u003cp\u003eTAS levels were measured in control, vehicle and DMBA groups. The TAS level in the DMBA-treated group was significantly lower than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Considering the TOS levels, the TOS level in the DMBA-treated group was significantly higher compared to control group, unlike the TAS level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) (Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eDetection of c-Abl and mTERT Localization In the DMBA-Treated Mouse Ovary\u003c/b\u003e\u003c/h2\u003e\u003cp\u003ec-Abl and mTERT protein localization were evaluated with immunofluorescence staining in the control and DMBA-treated mouse ovarian tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-A\u0026rsquo;, B-B\u0026rsquo;, C-C\u0026rsquo;). We determined that c-Abl and mTERT immunostaining patterns showed the same expression pattern as the control group and DMBA-induced ovotoxicity group. c-Abl and mTERT protein localizations were detected in the granulosa cells and oocyte in Graafian follicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA (A-A\u0026rsquo;, B-B\u0026rsquo;, C-C\u0026rsquo;)). In addition, we observed that mTERT immunostaining was more intense in the DMBA-treated group compared to the control group. The mTERT immunoflourescence intensity in both oocyte and granulosa cells was higher than that of c-Abl (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also examined the localization of c-Abl and mTERT in \u003cem\u003ein vitro\u003c/em\u003e mouse ovaries treated with short-term DMBA. We determined the expression of c-Abl and mTERT in both granulosa cells and oocyte in the developing follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA (A-A\u0026rsquo;, B-B\u0026rsquo;, C-C\u0026rsquo;)) and mTERT immunoflourescence intensity was higher in both granulosa and oocyte compared to c-Abl. In addition, mTERT expression in the DMBA-treated group was compared to the control group, both in the oocyte and in the granulosa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB and C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ec-Abl, mTERT, PCNA and Caspase Protein Expression in DMBA-Treated Mouse Ovary\u003c/h3\u003e\n\u003cp\u003eWe performed western blot experiment to compare protein expression levels between groups. As a result of our western blot experiment with ovarian tissues samples isolated from control, veichle and DMBA treatment groups, c-Abl expression decreased in ovarian tissues isolated from ovotoxicity group by applying \u003cem\u003ein vivo\u003c/em\u003e DMBA treatment (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA and B). We determined that mTERT protein expression level increased (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA and B). The PCNA protein level in the DMBA-treated ovary was significantly lower than the control group (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005) (Fig.\u0026nbsp;12A and B). There was no significant difference in Caspase protein level between experimental groups (Fig.\u0026nbsp;13A and B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ec-Abl\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003emTERT\u003c/b\u003e \u003cb\u003emRNA Expression In DMBA-Treated Mouse Ovary\u003c/b\u003e\u003c/p\u003e\u003cp\u003ec-Abl and mTERT mRNA expression levels were measured by qRT-PCR, and all were not changed in control and DMBA-treated groups (Fig.\u0026nbsp;14A and 14B).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDMBA is an organ-specific enviromntal carcinogen which has a potent negative effects on follicular and ovarian development in mammals (Ganesan \u0026amp; Keating \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), (Ganesan et al \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and cause ovotoxicity stimulate follicle loss by activating the primordial follicle population. Recent studies have also showed that this ovotoxicity can induce POI through dysfunctional primordial follicle activation (Sobinoff et al \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this study, we aimed to evaluate the possible roles of c-Abl and mTERT DMBA-induced ovotoxicity mechanism.\u003c/p\u003e\u003cp\u003eThe DMBA-treated ovaries presented the decrease in the number of primordial and primary follicles but the increase in the number of secondary follicles shows that DMBA stimulates follicle activation. However, the significantly increased number of atretic follicles in the DMBA-treated group was also remarkably evident. DMBA causes follicular atresia in the entire follicular stages and atretic follicles seen in the DMBA-treated ovaries are quite striking. When we examined the developing follicles in DMBA treated group, we observed deterioration in the cytoplasm and zona pellucida of oocytes compared to the control group. When we examined the nuclei of the oocytes in the atretic follicles of DMBA-induced group, we observed that there were errors in meiotic division. Our findings are consistent with the findings that DMBA exposure causes disruptions in the meiotic process (Song et al \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Yang et al \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We observed that both mitotic and apoptotic cells were activated in the granulosa cells in the follicles in the ovary treated by DMBA compared to the control group.\u003c/p\u003e\u003cp\u003eAMH expression begins in cuboidal granulosa cells of the primary follicles, which are formed after the primordial follicles are recruited from the follicle reserve as dormant follicle, and AMH level increases until the preantral and small antral follicular stages (Durlinger et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In the later follicular stages, AMH expression decreases and is not seen during follicular growth due to follicle stimulationg hormone (FSH). Also, AMH is expressed only in healthy follicles, not in follicles that undergo atresia (Durlinger et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Visser et al \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Decrease in serum AMH levels is associated with the reduction in the size of the antral follicle pool (de Vet et al \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, van Rooij et al \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In this study, we determined that the number of primordial follicles decreased in DMBA-treated mouse ovaries, whereas the number of primary and secondary follicles increased, in parallel, serum AMH levels increased. However, when we evaluated the structure of the developing follicles, we observed that DMBA causes impairment in cytoplasmic and nuclear maturation of oocytes. When we support these findings with the literature, we suggested that DMBA causes premature primordial follicle activation, increasing the number of developing follicles and AMH levels in DMBA-treated group.\u003c/p\u003e\u003cp\u003eThe TEM findings demonstrated that the number of transzonal projections decreased and their length was shortened in the DMBA-treated group, that the granulosa cells had damage in their mitochondria, and that they mostly lost their crystae structure. In addition, there were too many apoptotic granulosa cells in different stages developing follicles in the DMBA-treated group and errors occurred in the meiotic division of the oocyte, progressing from prophase I (PI) to metaphase I (MI). These findings show that premature follicle activation resulting from DMBA treatment negatively affects both oocyte maturation and the communication between granulosa cells and oocyte, causing impaired follicle development, triggering follicular atresia and leading a decrease in primordial follicle reserve. Based on this, we suggested that DMBA may adversely affect oocyte maturation and quality by inducing disruption of follicle development and meiotic division.\u003c/p\u003e\u003cp\u003eAntioxidant enzymes play an important role in defending free radical-mediated tissue or cellular damage (Nampoothiri et al \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). TAS level in ovotoxicity induced by DMBA was found to be lower than the control group. However, TOS level was found to be higher in the DMBA-treated group compared to the control group. As a result, we suggested that DMBA increases the oxidative stress level in the ovary and blocks antioxidant systems.\u003c/p\u003e\u003cp\u003eTelomerase activity and elongation of telomeres are necessary for proliferation of granulosa cells, especially in small and growing follicles (Liu \u0026amp; Li \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In a study, it was determined that estrogen positively regulates TERT gene expression by acting directly on the TERT gene promoter region and indirectly through the MYC gene (Liu \u0026amp; Li \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, it has been shown that estrogen receptor 1 and 2 are involved in the regulation of the transcription of the TERT gene in cancer and healthy differentiated cells (Grasselli et al \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Kondoh et al \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In our study, we determined an increase in the number of follicles in the secondary stage and a decrease in mTERT expression as a result of premature follicle activation in DMBA-induced ovotoxicity. Studies have shown that telomerase is short and telomerase activity is low in leukocytes and granulosa cells isolated from peripheral blood in women with POI (Butts et al \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Xu et al \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, telomeric shortening of granulosa cells and decreased telomerase activity in young women have been shown to cause ovarian failure (Butts et al \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This literature information confirms our datas. However, considering telomerase, germ cell growth and neoplastic immortalization, it is like a double-edged knife. It will be useful to observe how the proliferating granulosa cells will undergo a change with the effect of DMBA in the future with a longer exposure to DMBA.\u003c/p\u003e\u003cp\u003eWe determined that DMBA decreased its expression by inhibiting c-Abl in ovarian tissue treated with DMBA for one week. The fact that DMBA causes premature follicle activation and as a result of this, the increase in the number of follicles in the secondary stage shows us that oocytes and granulosa cells get rid of the repair mechanism due to DMBA suppressing c-Abl and they can continue their development by choosing an alternative pathway. The fact that there was no significant change in Caspase3 expression between the groups supports this finding. A study has shown that the carcinogenic effect of DMBA can be treated by applying the c-Abl inhibitor Imatinib (Morgan et al. 2013). In the light of the findings, we suggested that oocyte and granulosa cells escape from the DNA repair control mechanism by suppressing c-Abl after DMBA treatment and thus premature activated follicles develop into the secondary stage as the granulosa cells continue their proliferation.\u003c/p\u003e\u003cp\u003eIn our results; we showed that ovotoxicity caused by DMBA-treatment may cause premature depletion of developing follicles due to follicle activation. An increase in developing follicles is an increase in the total number of developed follicles. The increasing number of atretic follicles after DMBA-treatment presented disruption of communication between oocyte and granulosa cells, and damage to the nucleus during meiotic division. We showed that DMBA has an inhibitory effect on c-Abl expression in mouse ovary after 1 week of treatment, while it has a triggering effect on mTERT. We determined that c-Abl and mTERT have a potential role in DMBA-induced ovotoxicity. However, when the functional role of c-Abl and mTERT in ovotoxicity is investigated, we will have more detailed information about the molecular mechanisms.\u003c/p\u003e"},{"header":" MATERIAL AND METHODS","content":"\u003cp\u003e All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (Y\u0026Uuml;DETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. All experiments were performed in accordance with guidelines and regulations of Faculty of Medicine of Yeditepe University.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eGeneration of DMBA-treated mouse model\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eDMBA treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFemale BalbC mice (n\u0026thinsp;=\u0026thinsp;10 for each group) at 28 day of age, were obtained from the YUDETAM. A total of 4\u0026ndash;5 mice per cage were maintained for 14 days in a temperature-controlled environment under 12h:12h light-dark cycles with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. To induce ovotoxicity, mice were administered daily intraperitoneal injection (ip) of consecutive doses of either sesame oil containing vehicle control (\u0026lt;\u0026thinsp;10 \u0026micro;l/kg/daily sesame oil) or sesame oil containing DMBA 7,12-dimethylbenz(a)anthracene (DMBA) (Lgc standards; #DRE-C20745000) (1mg/kg/Daily dissolved in sesame oil) for 14 days (Borman et al \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMice were disected after 1 week from last treatment day. Right ovaries from each experimental group were used morphological evaluation (Hematoxylin and Eosin (H\u0026amp;E) staining and follicle counting (n\u0026thinsp;=\u0026thinsp;10), immunofluorescence staining and ultrastructural investigation) and left ovaries were used for western blot (n\u0026thinsp;=\u0026thinsp;5) and qRT-PCR experiment (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eDMBA treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the effect of DMBA \u003cem\u003ein vitro\u003c/em\u003e, the ovaries were dissected at 28 day and cultured in 6-well culture plates (n\u0026thinsp;=\u0026thinsp;6 for each group). Ovaries cultured with RPMI medium containing 10% FBS, 7,5 mg Ascorbic Acid, 1,25 ml L-Glutamine, 75 \u0026micro;l, 250 \u0026micro;l FGF (fibroblast growth factor), 30 \u0026micro;l LIF (Leukemia inhibitory factor), 37,7 \u0026micro;l SCF (stem cell factor), 3 ml B27 and penicillin/streptomycin for 4 days at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, culture media changes every 2 days. Ovaries were treated with control medium (no application), vehicle medium (0.01% acetone) and DMBA (50 nM dissolved in acetone) (Sobinoff et al \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTissue Processing and Hematoxylin and Eosin Staining\u003c/h2\u003e\u003cp\u003eAll ovarian tissue samples were fixed with 10% neutral buffered formalin for 18 hours, dehydrated using a graded series of 70, 80, 90 and 100% alcohol, vitrified in xylene and embedded in 60\u0026deg;C paraffin. 5\u0026micro;m serial sections were taken by microtome (Leica, #RM2245) onto poly-L-lysine coated slides (Thermo Scientific, #P4981). Paraffin sections were deparaffinized in xylene, rehydrated in alcohol series (100, 90, 80 and 70%). Sections were stained with H\u0026amp;E staining (Bio-optica Gill's hematoxylin; #06014/L, Bio-optica Eosin Y alcoholic solution; #10003/L). Then passed through the increased alcohol series, covered with mounting medium and examined under light microscope (Leica, #DM6000) (Yu et al \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFollicle counting\u003c/h2\u003e\u003cp\u003eThe number of follicles in all the serial sections of an in vivo and \u003cem\u003ein vitro\u003c/em\u003e ovaries were counted (Greenfeld et al \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). H\u0026amp;E staining was performed in serial sections to evaluate ovarian morphology and determine the number of primordial, primary, secondary, preantral and atretic follicles in the ovaries. 5 \u0026micro;m thickness sections were taken from paraffin blocks and the only follicles with visible nucleus seen in the oocyte were counted in each section. Follicles are different follicular stages were determined by counting from each group. After the total number of follicles was determined, the results of follicle counts between the groups were compared.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFor in vivo groups;\u003c/em\u003e primordial follicle was based on having an oocyte surrounded by a single layer of flat pregranulosa layer. Primary follicles were defined as an oocyte surrounded by a single layer of cuboidal granulosa cells. Secondary follicles were identified by presence of two or three layers of cuboidal granulosa cells surrounding the oocyte. It was defined by the emergence of small follicular spaces between granulosa cells in preantral follicles. Atretic follicle determined by a follicle that enters a degenerative process without ovulation (Greenfeld et al \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eFor in vitro groups;\u003c/em\u003e follicles with a clearly visible nucleus were counted. Primordial, growing and atretic follicles were determined for the \u003cem\u003ein vitro\u003c/em\u003e group. These follicles were classified as primordial follicles (flat pre-granulosa cells surrounding the oocyte), growing follicles (an enlarged oocyte surrounded by multiple cuboidal granulosa cells), and atretic follicles (a degenerate oocyte with collapse cytoplasm) (Zhang et al \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of Anti-M\u0026uuml;llerian Hormone (AMH) levels in DMBA-Treated mouse model serum.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter dissection, blood samples were obtained by cardiac puncture from mice all groups to measure the Anti-M\u0026uuml;llerian Hormone (AMH) levels. The blood samples were centrifuged at 4000 rpm for 15 minutes. The serum was isolated, frozen and kept at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. AMH level was measured by ELISA kit (Mouse AMH ELISA Kit, Sunred, #DZE201021297) according to the manufacturer\u0026rsquo;s instructions. Briefly, serum samples were centrifuged and then standards were prepared. The absorbance of each well was measured Microplate Spectrophotometer 450 nm wave length using a microplate reader through the Gen5 program.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eTransmission Electron Microscopy\u003c/h2\u003e\u003cp\u003eTransmission electron microscopy (TEM) was used to examine oocyte and follicle cells at the ultrastructural level ovarian tissues isolated from control (n\u0026thinsp;=\u0026thinsp;3), vehicle (n\u0026thinsp;=\u0026thinsp;3) and DMBA-treated group (n\u0026thinsp;=\u0026thinsp;3). Ovarian tissues were trimmed approximately 1mm\u003csup\u003e3\u003c/sup\u003e in size and fixed in 4% Gluteraldehyde (prepared in 0.1 M Sorensen\u0026rsquo;s Phosphate Buffer (SBP)) for 2 hours. Then, it was washed with SBP and fixed with osmium tetraoxide (OsO\u003csub\u003e4\u003c/sub\u003e) fixative prepared in 1% SPB. The tissues were then dehydrated. Subsequently, after incubating for 1 hour in 1% uranyl acetate solution prepared with 75% ethanol, 2 times for 10 minutes in Propylene oxide, 2 times in Propylene oxide + (Araldite master mix\u0026thinsp;+\u0026thinsp;2% accelerator BDMA) (1:1) mixture. Incubated at room temperature for 4 hours. Tissues that were incubated overnight in araldit were buried fresh araldite the next day and kept at 60\u003csup\u003eo\u003c/sup\u003eC for 48 hours. Sections taken from araldit blocks using ultramicrotome were visualized under a TEM microscope (ZEİSS-LEO 906E).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of Total Antioxidant Status (TAS) and Total Oxidant Status (TOS)\u003c/h2\u003e\u003cp\u003eTotal Antioxidant Status (TAS) and Total Oxidant Status (TOS) groups were performed using Rel Assay Diagnostic Kits. For TAS analysis, 30 seconds were waited and then measured with 660 nm wavelength. Reagent2 was then added and incubated for 5 min and measured with 660 nm wavelength. For TOS analysis, it was waited 30 seconds after adding reagent1 to serum samples and then measured with 530 nm wavelength. Then reagent2 was added and it was incubated for 5 min and measured with 530 nm wavelength. Measurements were made with Bio-tek SynergyTM HT Multi-Detection Microplate Reader device.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003e5 \u0026micro;m ovarian tissue sections were incubated in a 37\u0026deg;C incubator for 1h. Tissues were permeabilized and rehydrated with descending alcohol series and tissue sections were encircled with a histology pap pen to maintain staining solutions concentrated on the tissue during processing. Tissues were permeabilized in TBS with 0.1% Tween 20 (TBST), followed by antigen unmasking (Citrate Buffer) and blocking (TBS-T, NGS/5%) for 60 min at room temperature. After blocking tissues were incubated with c-Abl (PA5, 39688, Rabbit, Thermo, 1:100) primary antibody for 2 hours at room temperature. After washing with TBS-T, secondary antibody (Goat Anti-Rabbit IgG H\u0026amp;L (DyLight\u0026reg; 488) preadsorbed, Abcam, ab96883, 1:250) was placed for 1.5 hours at room temperature. Afterwards for double immunoflourescence staining, sections were blocked in and incubated mTERT (MA5-16034, Mouse mA6, Thermo, 1:100) primary antibody overnight at 4\u0026deg;C. The next day the sections were washed with TBS-T for 3 times for 5 minutes and incubated with secondary antibody (Goat Anti-Mouse IgG H\u0026amp;L Alexa Fluor\u0026reg; 647, Abcam (ab150115, 1:250) for 1,5 hour at room temperature. Sections were mounted with a DAPI mounting medium and visualized with Confocal microscope (Zeiss, LSM780) (Yaba et al \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e\u003cp\u003eAfter the samples were homogenized, protein was measured from tissue lysates. The total proteins were subjected to electrophoresis in %4\u0026ndash;12 Bis-Tris gels (ThermoFisher Scientific; NP0321) and transferred onto a PVDF membrane (ThermoFisher Scientific; IB401001) using the overnight wet transfer system. Subsequently, membranes were incubated overnight at 4\u0026deg;C with c-Abl (Sigma #A5844, 1:1000), mTERT (Santa Cruz #sc-7212, 1:1000), Caspase3 (D3R6Y, #14214S Cell signaling, 1:1000) and PCNA (D3H8P, #13110S Cell signaling, 1:1000) and Beta-actin (Cell Signaling #8H10D10, 15000) polyclonal antibodies. After incubation with primary antibody, membranes were washed 3 times for 5 minutes with TBS-T. Incubated with secondary antibody for 2 hours at room temperature and membranes viewed with CCD camera (Yaba et al \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e\u003cp\u003eThe total RNA was extracted from the \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e DMBA-treated ovarian tissues by using a Total RNA Purification Kit (Jena Bioscience) according to manufacturer\u0026rsquo;s procedure. The concentration and purity of the isolated total RNA was determined spectrophotometer (Bio-tek SynergyTM HT Multi-Detection Microplate Reader). Total RNA was reverse transcribed to cDNA with an SCRIPT cDNA Synthesis Kit (Jena Bioscience) according to manufacturer\u0026rsquo;s protocol with an OligodT primer. The target fragments were quantified by real-time PCR with a qPCR GreenMaster with UNG (Jena Bioscience) with 2\u0026micro;l of the cDNA template in a Real-Time PCR Detection System (Bio Rad). qRT-PCR was carried out using the following parameters: 5 min initial denaturation at 94 \u003csup\u003eo\u003c/sup\u003eC, followed by 30 cycles (amplification) at 92\u003csup\u003eo\u003c/sup\u003eC for 20 s, 59\u003csup\u003eo\u003c/sup\u003eC for 20 s, and 72\u003csup\u003eo\u003c/sup\u003eC for 1 min. Melt curve analyses were run with each series to confirm the specificity of the amplified products. The standard curves were prepared for c-Abl (F 5\u0026rsquo;-CGG GAC CAT GTT GGA GAT-3\u0026rsquo;, R 5\u0026rsquo;-TTC ATA CCG CAG CGA GATG-3\u0026rsquo;), mTERT (F 5'-CCTGCGGCCCATTGTGAAC-3', R 5'-GTG GAC TTG GCC TTG GCT ATC TCT-3') and Beta-actin (F 5\u0026rsquo;-GACCTCTATG- CAACACAGT-3\u0026rsquo;, R 5\u0026rsquo; -TTG CTG ATC CAC ATC TGCT-3\u0026rsquo;). Each sample was tested in duplicate. The gene expression datas were normalized to \u003cem\u003eβ-actin\u003c/em\u003e expression (Yaba et al \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Significant differences determined by two-way ANOVA test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor immunofluorescence intensity of c-Abl and mTERT expression and western blot results were analyzed by using ImageJ and statistical analysis was performed with GraphPad Prism. For the immunofluorescence intensity results data were analysed by Unpaired t test with Welch\u0026rsquo;s correction. For the WB results were analysed by a One-Way ANOVA test. For follicle counting results datas were analysed by Two-Way ANOVA test to compare multiple groups. For the qRT-PCR results were analysed by a Two-Way ANOVA test (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005).\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003ePOI:\u0026nbsp;\u003c/strong\u003ePremature Ovarian Infufficiency\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDMBA:\u003c/strong\u003e 7,12-Dimethylbenz[a]anthracene\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec-Abl\u003c/strong\u003e: Abelson Tyrosine Kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTERT:\u003c/strong\u003e Telomerase Catalytic Subunit\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E:\u003c/strong\u003e Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAMH:\u003c/strong\u003e Anti-Müllerian Hormone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEM:\u003c/strong\u003e Transmission electron microscopy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSBP:\u003c/strong\u003e Sorensen’s Phosphate Buffer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTAS:\u003c/strong\u003e Total Antioxidant Status\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTOS:\u003c/strong\u003e Total Oxidant Status\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP1:\u003c/strong\u003e Prophase 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMI:\u003c/strong\u003e Metaphase 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTZPs\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Transzonal Projections\u003c/p\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eAll animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. All procedures carried out in this study were conducted according to the Yeditepe University Experimental Animals Ethics Directive which has been prepared according to the rules and principles in Universal Declaration of Animal Rights,\u003c/p\u003e\n\u003c/div\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCorresponding author\u003c/h2\u003e\u003cp\u003eCorrespondence to Aylin Yaba.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding statement\u003c/h2\u003e\u003cp\u003eThis project supported by from The Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK#215S867).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE.Y performed western blot, ELISA, TAS/TOS. T.O. performed H\u0026amp;E staining, follicle counting and qRT-PCR. A.Y. performed TEM analysis, conceived and coordinated the study. A.Y, E.Y. and T.O. performed analysis of all experimental results and wrote the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis project supported by from The Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK#215S867).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data are within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBakalova R, Ohba H, Zhelev Z, Ishikawa M, Shinohara Y, Baba Y (2003) Cross-talk between Bcr-Abl tyrosine kinase, protein kinase C and telomerase-a potential reason for resistance to Glivec in chronic myelogenous leukaemia. Biochem Pharmacol 66:1879\u0026ndash;1884\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM et al (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. 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PLoS ONE 6:e21415\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang X, Zhang W, Wang Z, Zheng N, Yuan F et al (2022) Enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling. Cell Death Dis 13:87\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu J, Shore SK (1996) c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein. Mol Cell Biol 16:7054\u0026ndash;7062\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DMBA (7,12-dimethylbenz-[a]anthracene), Ovotoxicity, Mouse, c-Abl, mTERT","lastPublishedDoi":"10.21203/rs.3.rs-7571888/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7571888/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), is a potent carcinogen induces DNA damage and causes ovotoxicity with depletion of all follicle types in rat and mouse. c-Abl protein tyrosine kinase is activated by DNA double-strand breaks and proteins involved in the repair of lesions function in telomere control. Telomerase is one of the most important factor to limit telomeric shortening and maintain oocytes number in ovarian reserve. We showed before that mTERT associates directly with the c-Abl tyrosine kinase in mouse granulosa cells. Therefore, in the purposed study we aimed to show the mechanism of depletion of follicle reserve and atresia in the DMBA-induced ovotoxicity mouse model. In our study, we focused on the potential roles of c-Abl protein tyrosine kinase and mTERT telomerase catalytic subunit in ovarian follicles after DMBA exposure. We determined that exposure to DMBA resulted in decreased c-Abl tyrosine kinase activity in response to increased telomerase activity in mouse ovary. In conclusion, our results suggested that c-Abl and mTERT may have a functional role in folliculogenesis and mediate rapid depletion of ovarian follicles in DMBA-induced ovotoxicity mechanism.\u003c/p\u003e","manuscriptTitle":"Detection of c-Abl and mTERT Expression by DMBA-Induced Mouse Model of Premature Ovarian Insufficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 08:52:43","doi":"10.21203/rs.3.rs-7571888/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-21T06:22:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T07:23:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T09:52:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T15:39:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223949365202511106545397352161035727480","date":"2025-09-22T00:35:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310405300079388001312847507140314658099","date":"2025-09-16T17:27:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311013485490055499681790538334658634831","date":"2025-09-16T14:00:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-14T14:18:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-09T17:44:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T10:57:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Histology","date":"2025-09-09T09:06:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"074e075a-27ca-4e37-a4d3-69484afe0ede","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:08:45+00:00","versionOfRecord":{"articleIdentity":"rs-7571888","link":"https://doi.org/10.1007/s10735-025-10662-6","journal":{"identity":"journal-of-molecular-histology","isVorOnly":false,"title":"Journal of Molecular Histology"},"publishedOn":"2025-11-24 15:58:38","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-09-22 08:52:43","video":"","vorDoi":"10.1007/s10735-025-10662-6","vorDoiUrl":"https://doi.org/10.1007/s10735-025-10662-6","workflowStages":[]},"version":"v1","identity":"rs-7571888","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7571888","identity":"rs-7571888","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}