Cycloastragenol activation of Telomerase improves β-Klotho protein level and attenuates age-related malfunctioning in ovarian tissues

Age-related deterioration in the reproductive capacity of women is directly related to the poor developmental potential of ovarian follicles. Although telomerase plays a key role in female fertility, TERT-targeting therapeutic strategies for age-related female infertility have yet to be investigated. This study elucidated


INTRODUCTION
Impaired gamete development, a well-established ageing-related phenomenon, is the major etiological factor for infertility, miscarriage, and other pregnancy-related complications among aged women. The rapid advances in modern assisted reproductive technologies have not contributed to alleviating infertility among geriatric mothers. One of the major mechanisms of reproductive ageing is the degeneration of oocyte-surrounding somatic cells (Tatone and Amicarelli, 2013). Several studies have demonstrated that ovarian tissues and follicular microenvironment undergo marked changes during reproductive ageing (Pertynska-Marczewska and Diamanti-Kandarakis, 2016).
Although the genetic basis for ovarian ageing is known, most studies have focused on the free radical theory of ovarian ageing , Ito et al., 2008. Even though the role of critical proteins and signaling pathways in age-related female infertility is significant and has been investigated by many researchers (Ito et al., 2008, Briley et al., 2016, Lliberos et al., 2021, antiageing genes in ovarian tissues have gotten very little attention. Telomere length (TL) is a suitable biomarker for ageing, and TERT is a predominant determinant of TL (Vasilopoulos et al., 2019). TERT play critical functions in various tissues, and its dysfunction is associated with the impairment of tissue repair or regeneration in several pathologic conditions (Ito et al., 2008). Previous studies have reported that the highly conserved telomeric DNA sequence (TTAGGG)n is maintained through telomerase activation and telomerase dysfunction leads to morphological, biochemical, and functional changes in the tissues (Vasilopoulos et al., 2019).
Transgenic expression of TERT promotes the proliferation of primary cell culture and enhances the reprogramming efficiency of induced pluripotent stem cells (Hidema et al., 2016). In addition to J o u r n a l P r e -p r o o f maintaining TL, TERT is reported to be involved in various cellular functions (Martínez and Blasco, 2011, Vasilopoulos et al., 2019, Butts et al., 2009, Treff et al., 2011. Female infertility results from shortened TL in various reproductive tissues (Ito et al., 2008). The downregulation of TERT mRNA and telomerase activity in oocyte-surrounding granulosa cells (GCs) is one of the major mechanisms involved in female gamete deterioration (Tománek et al., 2008, Lavranos et al., 1999, Kosebent et al., 2018. According to reports, TERT is said to have a role in a number of cellular processes in addition to TL maintenance (Butts et al., 2009). Several proteins are involved in TERT transcription and activation (Yuan et al., 2019). Conversely, TERT is reported to regulate various proteins (Xu et al., 2021). A previous study identified the correlation between Klotho (KL) and TERT and demonstrated that the downregulation of KL impairs telomerase activity in stem cells (Park et al., 2009). The silencing of KL, results in various pre-mature ageing symptoms and decreases the lifespan of mutant mice (Jung et al., 2017). KLB, a subfamily of KL, exerts anti-ageing effects by forming a complex with FGFR1 and facilitating FGF21 signaling (Suku et al., 2020, Fan and Sun, 2016, Kim et al., 2011, Yan et al., 2021, Yie et al., 2012. Furthermore, the FGFR1/KLB complex promotes gonadotropin-releasing hormone (GnRH) secretion in the pituitary gland and regulates the onset of puberty (Misrahi, 2017). Moreover, KLB can individually regulate the onset of female puberty (Misrahi, 2017. Ovary-specific KLB knockout significantly alters cyclicity, downregulates luteinizing hormone secretion, and consequently decreases female fertility .
The above studies suggest that telomerase activation may positively affect fertility. Telomerase activation or telomerase-based therapies have been developed to improve health span, and reduce age-related diseases (Relitti et al., 2020, Rafat et al., 2022, Blasco and Bär, 2016, Jaijyan et al., 2022. In traditional Chinese medicine, Astragalus membranaceus root extracts has been used as an antiaging compound. Cycloastragenol (CAG) is triterpenoid saponin and an active ingredient of Astragalus membranaceus root (Szabo, 2014, Yu et al., 2018a. CAG was previously been used to treat diabetes, reduce inflammation, and treat macular degeneration (Dow and Harley, 2016, Zhu et al., 2021, Zhang et al., 2020. Anti-cancer and neuroprotective properties of CAG have also been discovered by researchers (Hwang et al., 2019, Li et al., 2020. One of the exciting properties of CAG is telomerase activation, enhancing the protein level of TERT, and improving the telomere length (Yılmaz et al., 2022, Hong et al., 2021. However, it is unknown how CAG could affect female fertility, particularly in ageing conditions. Therefore, the present study hypothesized that CAG may postpone female fecundity by upregulating TERT and KLB.

J o u r n a l P r e -p r o o f
Age-related infertility is one of the leading factors affecting a woman's ability to conceive and deliver a healthy child. The above literature suggest that telomerase and KLB are crucial for female fertility and may have some connections. We investigated both the two anti-ageing processes therefore TERT and FGF21/FGFR1/KLB pathway in human and mouse GCs and in mouse ovarian tissues. We noticed a drastic reduction in the protein levels of TERT and KLB in D-galactose (Dgal)-induced ovarian ageing model and solo activation of TERT recovered the FGFR1/KLB pathway and ovarian follicles. Furthermore, Doxorubicin (DOX) which dose dependently alter ovarian hormones and inhibit follicular formation. CAG mediated Telomerase activation and recovery of FGFR1/KLB pathway, not only improved the hormonal level, but also recovered the number of developing follicles in ovary.

MATERIAL AND METHODS
All experiments were performed using female ICR mice (bodyweight, 20-25 g; aged 12-14 weeks), which were maintained at the Animal Care Facility of the Gyeongsang National University Institute of Animal Care Committee (GNU-130902-A0059). The animal experiments were performed according to the Code of Practice for Care and Use of Animals for Experimental Purposes.
Chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise stated.

Experimental design
Primary mouse GCs were collected from mouse ovaries and cultured in the presence or absence of CAG for 24 and 48 h and analyzed the gene expressions and protein levels of Tert and Klb. Next, to develop an ovarian-ageing model and to explore the influence of ageing on GCs, D-gal was given to Human GCs (COV434 cell line). The effective concentration of CAG for mitigating D-gal-induced toxicity in COV434 cells was determined using the MTT assay. Furthermore, Tert was inhibited via Doxorubicin (DOX), and Knockdown via siRNA and KLB protein was analyzed. Moreover, cycloastragenol (CAG catalog no. SMB00372) was treated to DOX-treated and D-gal-treated granulosa cells and KLB and other ovarian follicular growth related proteins (ER-α, SF-1, FOXL2, ERK1/2, PI3K, AKT, and mTOR) were examined using immunofluorescence and western blotting.
ICR strain female mice were used in two investigations. In the first experiment, the female mice aged 8-10 weeks were randomly assigned to one of three groups: saline, D-gal, or D-gal + CAG . The mice in the D-gal + CAG group were given D-gal intraperitoneally for 15 J o u r n a l P r e -p r o o f days, then CAG (20 mg/kg bodyweight/d) intraperitoneally for 28 days in combination to D-gal.
Mice in the control group were intraperitoneally administered with an equal volume of saline. In the second experiment the female mice were randomly divided into the following three groups: saline, DOX, and DOX + CAG groups. In both the experiments the ovarian follicles were examined.
Furthermore, follicles and granulosa cells specific proteins, and TERT and KLB/FGFR1proteins were examined via western blot and immunofluorescence.

DOX and CAG treatment
ICR strain of female mice aged 8-10 weeks were randomly divided into the following three groups (10 mice per group): saline, DOX, and DOX + CAG groups. DOX was intraperitoneally administered at a dose of 20 mg/kg of mice for 7 days (20 mg/kg bodyweight; equivalent to a dose of 6.5 mg/m 2 in patients) ( Figure S1A) .

D-Gal and CAG treatment
ICR strain of female mice aged 8-10 weeks were randomly divided into the following three groups (10 mice/group): saline, D-gal, and D-gal + CAG groups . D-gal (200 mg/kg bodyweight/day) was intraperitoneally administered on alternate days for 42 days. Mice in the control group were intraperitoneally administered with an equal volume of saline. In the D-gal + CAG group, the mice were intraperitoneally injected with D-gal for 15 days, followed by intraperitoneal administration of CAG (20 mg/kg bodyweight/d) for 28 days along with D-gal ( Figure S1B). The protein levels of FGFR1, ESR1, p-MAPK1/3, p-AKT1, and p-MTOR were analyzed via immunofluorescence and western blot.

Primary mouse GC (pGC) culture
Mouse pGCs were obtained from the ovaries of euthanized females. Female mice were injected with 5 IU of pregnant mare serum gonadotropin (Daesung Microbiological Labs, Gyeonggi do, Republic of Korea) and the ovaries were collected in L-15 Leibovitz-Glutamax medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) after 72 h. Cumulus oocyte complexes (COCs) containing immature oocytes in the antral follicles from both the outer (ovarian surface) and inner J o u r n a l P r e -p r o o f (deep ovarian tissue) layers of the ovarian cortex were isolated. Mouse pGCs were obtained from COCs and cultured in previously described media and conditions (Baufeld and Vanselow, 2013).

Human GC line culture and MTT assay
The human GC line COV434 was a kind gift from Professor Jeehyeon Bae (Chung-Ang University, Seoul, Republic of Korea). The cell line was cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37.0 °C and 5% CO 2 . The viability of cells was examined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, following the manufacturer's instructions (Sigma). Briefly, COV434 cells were cultured in the wells of a 96-well plate (0.8 × 10 4 per well; 60% confluent cells) containing 200 μL DMEM for 24 h. The medium was replaced with fresh medium containing various concentrations of CAG and the cells were incubated for 24 h. The cells were then incubated with MTT solution for 3 h. Next, 100 µL dimethyl sulfoxide was added to the wells and the samples were agitated for 10-20 min on a shaker.
The absorbance of the mixture at 550-570 nm (L1; absorbance of live cells) and 620-650 nm (L2; absorbance of cell debris) was examined using a microplate reader.

Female embryo gonad culture and treatment
Female mouse fetal gonads were cultured as described previously (Morohaku et al., 2016). Briefly, fetal mouse gonads obtained at day 12.5 post-coitum were separated from the mesonephros and cultured in Transwell-COL membranes (Sigma cat. # CLS3491) for 23 days. The basal medium was alpha-minimal essential medium (Gibco, Thermo Fisher Scientific) supplemented with 1.5 mM 2-Oα-D glucopyranosyl-L-ascorbic acid (Tokyo Chemical Industry, Tokyo, Japan), 10 U/mL penicillin, and 10 µg/mL streptomycin (Sigma-Aldrich), FBS (Gibco, Thermo Fisher Scientific), serum protein substitute (SAGE In-Vitro Fertilization), and β-estradiol (Santa Cruz Biotechnology, Dallas, Texas, USA). The samples were treated with the estrogen receptor antagonist ICI 182780 (cat. # 1047, Tocris Bioscience, Bristol, United Kingdom) at a concentration of 10 µM from day 5 to day 11. On day 24 of culture, the gonads were fixed in 4% formaldehyde and stored at 4 °C until analysis. The mRNA was extracted using an RNA isolation kit (PicoPure, Arcturus, Thermo Fisher Scientific), following the manufacturer's instructions. The concentration of mRNA was examined by measuring the absorbance at 260 nm using Nanodrop. The mRNA was reverse-transcribed into first-strand cDNA using iScript reverse transcriptase (Bio-Rad, Hercules, CA, USA). The primers for qRT-PCR analysis were designed using the National Center for Biotechnology Information nucleotide database and Primer3 (v. 0.4.0) software and are listed in Table 1.

Modeling of hTERT and molecular docking
The homology modeling based in silico study aimed to predict the binding mode of Cycloastragenol (CAG) inside the active site of human telomerase. According to the literature review, only the structure of Tribolium castaneum telomerase is currently available in Protein Data Bank (PDB) (Baginski and Serbakowska, 2020). The first human telomerase modeled structure reveals that it's a multidomain enzyme and its catalytic domain found in telomerase reverse transcriptase (TERT) (Steczkiewicz et al., 2011). We therefore, modeled human TERT (hTERT) domain using Tribolium castaneum inhibitor bound structure PDB id 6E53 as a template (Hernandez-Sanchez et al., 2019).
The sequence of hTERT was obtained from UniProt (https://www.uniprot.org/) and aligned to the J o u r n a l P r e -p r o o f Tribolium castaneum TERT structure in Discovery Studio (DS) v18 (www.accelrys.com Accelrys Inc. San Diego, USA). Following that, the Build Homology Model protocol available in DS was utilized to build the hTERT model. In addition, two well-known modeling servers (Swiss-Model and I-TASSER) were utilized to model TERT for comparative analysis and model selection. The Swiss-Model performs template-based modeling whereas I-TASSER uses threading or multiple templates based approach (Roy et al., 2010). To select the final model the best model from each approach was validated using PROCHECK using PDBsum and ProSA webserver Sippl, 2007a, (Laskowski et al., 1997a). The final model was then energy minimized and prepared using Clean Protein protocol of DS for molecular docking with CAG. Due to the lack of binding site information between hTERT and CAG we utilized CB-Dock2 web server for the identification of potential cavities on hTERT(Y. . Both hTERT and CAG were submitted to the server and the potential cavities were detected. Subsequently the blind docking was also performed by server using Auto Dock vina (Trott and Olson, 2010). The CB-Dock2 predicted docking sites and affinities were further validated using another docking run in Genetic Optimization of Ligand Docking (GOLD v5.2.2) program (Marcel L. . For comparison we additionally docked CAG on known TERT active site. The binding site was defined using the template superimposition within the 9Å radius in DS. A total of fifty conformers were generated for each binding cavity and ranked according to GOLD scoring function (Sapundzhi et al., 2019).

Molecular dynamics (MD) simulations
MD simulations were performed to further validate the hTERT-CAG complex under physiological conditions using Groningen Machine for Chemical Simulations (v5.1.5). The simulation parameters for the hTERT model and CAG were generated using the CHARMm27 force field and Swiss Param, respectively. The simulation box was hydrated with the TIP3P water model, and the simulation system was neutralized using 52 Cl − ions. Subsequently, energy minimization and equilibration were performed. Finally, the simulation was performed under periodic boundary conditions. The detailed procedure used is summarized elsewhere. Finally, the MD simulation trajectory was used to calculate the binding free energy (ΔG) of the hTERT-CAG complex using the molecular mechanics Poisson-Boltzmann surface area, a computationally rigorous method. In total, 40 frames were generated from the simulation trajectories. The final binding free energy was calculated as follows: J o u r n a l P r e -p r o o f The final ΔG bind value for the hTERT-CAG complex was the average value from 40 to 50 ns of the MD simulation trajectories.

Immunoblotting
The ovaries were freshly collected from mice after treatment, frozen in liquid nitrogen, and stored at −80 °C. The tissues were then homogenized in a protein extraction solution pro-prep™ (iNtRON Biotechnology, Burlington, NJ, USA, cat. # 17081), following the manufacturer's instructions (iNtRON Biotech, Inc.) using a homogenizer . The tissue lysate (protein homogenate) was centrifuged at 13200 rpm at a controlled temperature (4 °C) for 25 min. The proteins (supernatants) were collected and stored at −80 °C for further analysis. The concentrations of the protein were determined using the Bradford assay (Bio-Rad Laboratories Hercules, CA, USA, cat. # 5000002), following the manufacturer's instructions. Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% or 12% gel. The resolved proteins were then transferred to polyvinylidene difluoride (Sigma-Aldrich, cat. # GE 10600023) membrane. The membrane was blocked with blocking solution (5% skim milk) for 1 h. Next, the membrane was incubated with the primary antibodies at 4 °C overnight, followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 90 min. After washing thrice with PBST for 10 min, immunoreactive signals were detected using Super Signal™ West Femto Maximum Sensitivity Substrate (Thermo cat. # 34095) and analyzed using the iBright™ FL1500 Imaging System (Thermo Fisher cat. # A44115). A protein ladder (Abcam, USA, cat. # ab116029) was used to detect proteins based on their molecular weights. ImageJ software (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij) program was used to detect the optical densities of bands.

Ovarian histology and follicle counting
Mice were sacrificed after the drug treatment to perform morphological analysis and follicle counting. The ovaries were fixed in 4% ice-cold paraformaldehyde in phosphate-buffered saline (PBS) for 12 h and fixed with 4% paraformaldehyde for 48 h and 20% sucrose solution for 72 h. The ovaries were then embedded in optimal cutting temperature compound and stored at −80 °C. The ovary sections (12 μM) were prepared on plus-charged slides using a Leica cryostat (CM 3050C, Germany). The follicles in the ovaries were counted according to a previously described method (Bernal et al., 2010). Briefly, the number of primordial follicles was counted in all serial sections of J o u r n a l P r e -p r o o f the ovary. The follicles were classified based on the GC layers. The primordial follicle has one layer of flattened granulosa surrounding the oocyte. The primary follicle has two layers of cuboidal GCs, while the secondary follicle has more than two layers of cuboidal GCs surrounding the follicles. The antral follicle was easily classified because it contained one or more antral spaces and multiple layers of cuboidal GC layers (Borgeest et al., 2002).

Immunofluorescence
Tissue sections or cultured cells [4 chamber culture slides (cat. No. 154917PK) fixed in 4% formaldehyde] were subjected to immunofluorescence staining as previously described . Briefly, the slides containing samples were rinsed thrice with 0.3% polyvinyl alcohol ( The images were captured using a confocal laser-scanning microscope (Fluoview FV 1000, Olympus, Japan) after washing the samples thrice. The relative integrated density of the signals was measured using the ImageJ analysis program (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij) software.

Probe
The human telomeric probe containing the telomeric DNA sequence TTAGGG was simultaneously amplified and labeled with digoxigenin (dig) coupled to dUTP using PCR with human genomic DNA as the template, 5-(CCCTAA) 7 -3′ primer, and a dig-labeling kit (Roche, Mannheim, Germany).
Quantitative FISH analysis was performed following the previous protocol with minor modifications (Sohn et al., 2012). Briefly, the cultured cells were incubated in RNase A (Sigma) and dehydrated using an increasing ethanol gradient. Dig-labeled probes containing hybridization solution (Roche) were dropped onto the slides and the samples were denatured at 78 °C for 10 min and hybridized at J o u r n a l P r e -p r o o f under a fluorescence microscope (Model AX-70, Olympus, Tokyo, Japan) at green (FITC) and red (PI) dual excitation wavelengths. The images were captured using a digital camera Olympus) and analyzed using MetaMorph  (Universal Imaging Co., PA, USA), an image analysis program.
Telomere-specific signals in at least 100 interphase nuclei were examined for each specimen.

Telomerase Activity
Primary cultured mice granulosa cells (isolated form mice ovarian follicles) were treated with CAG for 24 h. Quantitative determination of mouse telomerase concentrations was performed in plasma preparations using a colorimetric sandwich-ELISA assay, Mouse TE(telomerase) ELISA Kit (catalog no.E-EL-M1125 Elabsciences®, Houston, TX, USA. The optical density (OD) was measured at 450 ± 2 nm.

Statistical analysis
All data were statistically analyzed using the GraphPad Prism software (version 6.0 for Windows; GraphPad Software, San Diego, California, USA; www.graphpad.com). The comparison of the mean values between multiple groups was performed by the analysis of variance (ANOVA) followed by Sidak's multiple comparison test and Tukey's multiple comparison test. For all J o u r n a l P r e -p r o o f comparisons, p values < 0.05 was considered statistically significant and numerical data were expressed as the mean ± SD.

Ageing dysregulates the Klb and Tert protein levels in cultured pGCs
To initiate our experiment, we qualitatively examined the protein levels of KLB and FGFR1 in a human granulosa cell line (COV434) and Klb and Fgfr1 proteins in mouse ovaries (Fig. 1A). We hypothesized that KLB gene is Telomerase dependent in granulosa cells and to activate telomerase we used small molecule cycloastragenol (CAG) (Ip et al., 2014). In order to determine the effective concentration of CAG, the COV434 cell line was treated with D-gal and various concentrations of CAG (1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 7.5, and 10.0 µM), and D-gal-induced toxicity was assessed via MTT assay (Fig. S2A). D-gal-induced cytotoxicity against GCs was considerably reduced by 2.5 µM of CAG treatment. Since previous research has shown a connection between prolonged pGCs culture and the induction of ageing-like phenotypes (Kulus et al., 2020), we next assessed the effects of CAG on pGCs cultured for 24 and 96 h. Intriguingly, CAG mitigated the downregulation of the anti-ageing proteins Tert and Klb in prolong cultured pGCs (Fig. 1B).
FGF21 is the ligand of FGFR1/KLB co-receptors and its treatment to granulosa cells enhances estradiol production via AKT/mTOR signaling. We supplied fgf21 to the primary cultured mouse granulosa cells and examined AKT and mTOR via western blot. Interestingly, the levels of p-Akt1 and p-mTOR proteins did not differ significantly between the control and FGF21-treated samples, but were markedly increased (p < 0.05) after CAG treatment (Fig. 1C). After obtaining the aforementioned data, we argued that if CAG increases the FGFR1/KLB downstream markers, it may be because Tert is activated. Furthermore, if TERT is involved, than its protein level should be directly proportional to KLB. Using immunofluorescence, we measured the amounts of TERT and KLB proteins in control and CAG-treated pGCs grown at 24 h and 96 h (Fig. 1D). The protein levels of Tert and Klb in CAG-treated pGCs were significantly (p < 0.05) higher than that in control pGCs.
Next, we analyzed the telomerase activity in primary culture granulosa cells using CAG (Fig. 1E).
The results showed significant (p < 0.05) enhancement in the Telomerase activity in the presence of CAG. Even more, the telomere length was examined in 24 h and 96 h cultured control and CAG treated pGCs using FISH with a telomere-specific DNA probe (Fig. 1F).

J o u r n a l P r e -p r o o f
Previous research has shown that DOX dose dependently alters ovarian hormone and inhibits follicular growth (Aziz et al., 2020). To clarify the importance of KLB for ovarian functioning and its link with telomerase, COV434 cells were treated with DOX. Confocal microscopy analysis revealed that DOX significantly (p < 0.05) downregulated the protein level of KLB but not that of FGFR1 ( Fig. 2A). Next, the DOX-treated GCs were transfected with the hTERT plasmid, which resulted in the upregulation of KLB (Fig. 2B). However, neither the control-treated nor the DOXtreated GCs' TERT protein levels were significantly changed by KLB plasmid transfection (Fig. 2C).
In contrast, CAG significantly increased the TERT protein.
We then investigated the effect of D-gal on granulosa cells by examining the TERT and KLB protein levels. We treated the COV434 granulosa cell line with D-gal and added CAG (2.5 µM for 24 h).
TERT plasmid was also transfected to the D-gal-treated granulosa cells. Both the CAG treatment and TERT plasmid transfection significantly (p < 0.05) reduced the RAGE (main marker of ageing) level in granulosa cells (Fig. 2D). Next, the mRNA level of Telomerase RNA Component (hTR), and TERT were examined in the D-gal, and CAG co-treated samples (Fig. S2B). Which also showed that CAG significantly (p < 0.05) retained the TERT gene expression in the CAG co-treated groups as compared to control, and D-gal groups. Next, the effect of D-gal on TERT and KLB was examined in D-gal-treated COV434 cells (Fig. 2E). We observed that treatment with CAG markedly mitigated the D-gal-induced downregulation of TERT and KLB. Moreover, the enhanced TERT protein level was also detected in the CAG treated group. To gain a more in-depth picture regarding the GCs functioning, we examined estrogen receptor 1 (ER-α), and Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1) (Fig. S2C). The results demonstrate that CAG dramatically increased both female hormone-regulating proteins, even when co-treated with D-gal. To clarify whether CAG activated Telomerase is responsible for KLB enhanced expression, TERT was knocked down via siRNA and CAG was treated to the GCs (Fig. 2F). The gene expression of KLB was downregulated even in the presence of CAG. The results were further confirmed by a western blot analysis of KLB protein (Fig. 2G). Furthermore, the telomere-specific DNA Probe was used to examine the telomere length in the D-gal treated, DOX-treated, and CAG co-treated GCs using Fluorescence In situ hybridization (Fig. S2D). The results showed that CAG significantly mitigated the DOX-induced and D-gal-induced downregulation of telomerase protein level and telomere length.

J o u r n a l P r e -p r o o f
The TERT is catalytic subunit of human telomerase protein, which comprise of TEN (telomerase essential N-terminal domain), TRBD (telomerase RNA binding domain), RT (reverse transcriptase domain) and CTE domains (C-terminal extension) (Fig. 3A). The BLASTp template search revealed that sequence alignment of human TERT and Tribolium castaneum PDB 6E53 showed 26.82% identity (Fig. 3B) and E value 3e -13 . As a result, the available X-ray structures of T. castaneum structures were analyzed and inhibitor bound structure of T. castaneum (PDB is 6E53) was selected as a template for modeling of hTERT (Fig. 3C) (Hernandez-Sanchez et al., 2019). In this work, hTERT catalytic subunit (excluding TEN domain) was modeled. The aligned sequence file of hTERT and template was subjected to Build Homology Model protocol in DS (Fig. 3D) (Chen et al., 2017). The modeler produced 10 models and ranked them according to probability density function (PDF) total energy and discrete optimized potential energy (DOPE) score ( Table S1). The model Human.M0001 displaying lowest PDF total energy and DOPE score was selected initially (Fig. 3D).
The quality of the model was assessed using superimposition of the on the template which showed acceptable root mean square deviation (Fig. 3F). Additionally, for the selection of better model, further modeling was carried out with Swiss-Model and I-TASSER server (Fig. S3). The best model from each approach was selected and superimposed on each other, revealed all the models adopt similar confirmations (Fig. S3A-D). Additionally the models were validated using PDBsum webserver for Ramachandran Plot analysis which analyses the stereochemical properties of the three-dimensional structure (Laskowski et al., 1997b, Wiederstein andSippl, 2007b).
Ramachandran plot analysis revealed that model from DS displayed 96.1% residues are in allowed region this is followed by Swiss-Model model and I-TASSER model ( Fig. 3F and Table S2). The detailed validation of model demonstrated that model obtained from DS could be considered for further studies.

Molecular docking and MD simulations
Molecular docking approach was used to predict the binding mode of CAG with hTERT. Initial docking sites were predicted using CB-Dock2 (Y. . The hTERT and CAG both were provided as an input for the prediction of cavities on hTERT (Fig. S4A-B). The three top predicted sites were subsequently docked using CB-Dock2 and the docking score, molecular interactions were observed (Table S3). It is noteworthy to mention that Cavity1 predicted by CD-Dock2 lies in the close proximity to the inhibitor binding site (Fig. S4B). The CB-Dock2 results were further validated using second docking run in GOLD program (M L Verdonk et al., 2003). A total of 50 conformers of CAG were generated for each predicted site and inhibitor binding site as well. The best conformer J o u r n a l P r e -p r o o f was selected from largest cluster which showed acceptable docking Goldscore and desirable molecular interaction with residues of hTERT (Sapundzhi et al., 2019). Both the docking runs indicates that CAG displayed highest affinity towards Cavity1 with vina score -8.1 kcal/mol and Goldscore of 55.17, this is followed by Cavity3 and Cavity2 (Table S3). Interestingly the inhibitor binding site displayed a lower Goldscore of 44.64 when compared with Goldscore of Cavity1, 55.17.
This indicates that Cavity1 maybe the binding site of CAG on hTERT (Fig. S4C-E).
The selected binding conformation of CAG-hTERT from Cavity1 was further simulated under physiological conditions. The main objective of the molecular simulations was to calculate the binding affinity of the CAG towards hTERT using computationally exhaustive methodology. The stability of the 50ns simulation trajectories were analyzed for root mean square deviation and fluctuations (RMSD and RMSF) . The analysis revealed that the CAG-hTERT complex displayed stable RMSD and acceptable RMSF fluctuations ( Fig. 4A-B). Further, the binding free energy analysis of last 10ns stable simulation trajectory revealed that CAG could bind with good reasonable binding affinity (-101.67 kJ/mol) with the hTERT protein (Fig. 4C). The per residue contribution of analysis of CAG-hTERT complex revealed that residues Val596, Lys626, Asn899, and Arg901 can contribute significantly via hydrophobic interactions (Fig. 4D). The binding mode of the hTERT-CAG complex was studied using the average structure from last 5ns simulation trajectory (Fig. 4E). It can be seen from Fig. 4F that CAG forms three hydrogen bonds with Leu630, Val897 and Leu900 of hTERT. Additionally, CAG also forms van der Waals interactions with Gly588, Gly629, Arg631, Asp628, Val898, Asn899, Arg901 and π-alkyl interactions with Val596, Lys626 and Cys896 (Fig. 4G).

CAG-activated Telomerase protects ovarian follicles against D-gal-induced ovarian ageing
Previous studies have reported that D-gal adversely affects female reproduction and promotes premature follicular loss (Wang et al., 2019). To understand the effect of D-gal on TERT and FGFR1/KLB signaling, fertile female mice were treated with D-gal and D-gal + CAG and their ovarian morphology was examined using hematoxylin and eosin (H&E) staining (Fig. 5A). H&E staining revealed that CAG significantly (p < 0.05) mitigated D-gal-induced follicular loss. Next, the protein levels of several key GC function-related proteins, such as Esr1, Nr5a1, and Foxl2, were examined using western blotting (Fig. 5B). Treatment with CAG mitigated the D-gal-induced downregulation of Esr1, Nr5a1, and Foxl2 protein levels. Similarly, treatment with CAG upregulated the Klb and its downstream signaling proteins, such as ERK1/3 and PI3K (Fig. 5C). Next, the effects J o u r n a l P r e -p r o o f of D-gal and CAG on female mouse embryonic gonads was analyzed. The gonads obtained from 12.5-day female mouse embryos were cultured for 23 days and treated with D-gal and D-gal + CAG (Fig. 5D). Treatment with D-gal significantly (p < 0.05) inhibited follicle formation and decreased gonad size, while CAG alleviated D-gal-induced ovarian toxicity. Furthermore, Klb protein was also significantly (p < 0.05) downregulated in cultured gonads, whereas that of Fgfr1 was not significantly affected upon treatment with D-gal (Fig. 5E). Treatment with CAG mitigated the Dgal-induced downregulation of Klb, impaired follicle development, and decreased gonad size.

CAG ameliorates DOX induced suppression of TERT and KLB and diminishes ovarian follicles in mouse ovaries
DOX alter ovarian hormone secretion and regulation by inhibiting Follicular development in the ovaries of fertile female mice treated with the DOX and CAG was examined (Fig. 6A). DOX also alter female hormonal regulation which is may be due to reduction in Klb expression. We found that DOX markedly impaired follicular development and significantly (p < 0.05) downregulated the Klb protein in mouse ovaries (Fig. 6B). However, treatment with CAG mitigated DOX-induced impaired follicular development and downregulation of Klb. Furthermore, it has been previously reported that ageing ovarian tissues, especially GCs, are characterized by mitochondrial dysfunction (Ernst and Lykke-Hartmann, 2018). DOX disrupts mitochondrial functioning and compel them to produce more ROS . Since TERT is involved in mitochondrial functioning and mitochondrial DNA maintenance, we looked at the mitochondrial markers Cytochrome C and Bcl2 (Fig. 6C).
Treatment with DOX markedly decreased Bcl2 and significantly increased Cyto C protein levels. In contrast, treatment with CAG mitigated DOX-induced Bcl2 downregulation and Cyto C upregulation. While Bcl2 and Cyto C protein levels were not significantly (p < 0.05) different between the CAG-treated and control groups. High protein level of Cyto C initiate apoptosis and thus we checked the protein level of Casp-3 via western blotting (Fig. 6D). Compared with those in the DOX-treated group, the ovarian protein level of Casp3 was significantly downregulated in the CAG-treated group. Nrf2-antioxidant response element pathway-related proteins block mitochondria initiated apoptosis. We found that the protein levels of Nrf2 and HO-1 were significantly (p < 0.05) upregulated, whereas those of Keap1 were significantly (p < 0.05) downregulated (Fig. 6E). These results suggest that CAG-activated Tert mitigates DOX-induced ovarian toxicity.

J o u r n a l P r e -p r o o f
Despite rapid advancements in assisted reproductive technology, there has not been significant success in the discovery of effective treatments for age-related female infertility. This study examined the role of the anti-ageing proteins TERT and KLB in mitigating ovarian ageing. Agerelated ovarian dysfunction is associated with the downregulation of TERT, which has significant effects on the FGFR1/KLB signaling pathway. We investigated the TERT activation effects on the FGFR1/KLB co-receptor pathway and its downstream signaling in granulosa cells and in whole mouse ovaries. We found that D-gal promoted ovarian ageing by dysregulating TERT and increasing FGF21 resistance, which can be attributed to the downregulation of KLB. CAG-activated TERT significantly upregulated the protein level of the FGFR1/KLB in D-gal induced ovarian ageing model. These findings are consistent with the role of telomerase and KLB in female fertility (Xu et al., 2020, Liu andLi, 2010). However, further studies are needed to establish the correlation between TERT and the FGFR1/KLB signaling pathway and the alleviation of age-related follicle depletion in aged females.
Several telomerase-based therapies have been developed to improve the health span, enhance longevity, and reduce mortality (Relitti et al., 2020, Rafat et al., 2022, Blasco and Bär, 2016, Jaijyan et al., 2022. Additionally, various therapeutic strategies, such as activation of telomerase, transfection of telomerase sequences, and reactivation of silenced telomerase, have been employed (Jaijyan et al., 2022, Bernardes de Jesus et al., 2012. Compared with other methods, treatment with pharmacological telomerase activators is a better therapeutic strategy owing to the easy regulation of treatment duration and dosage effects (Harley et al., 2011, Bernardes de Jesus et al., 2012. The activity of CAG, a potent telomerase activator, has been demonstrated in immune cells, neonatal keratinocytes, and fibroblasts in culture (Harley et al., 2011). Several in vivo studies have demonstrated that CAG increases the TL in leukocytes (Harley et al., 2011, Salvador et al., 2016. Similarly, dietary supplementation of CAG in mice promoted the elongation of critically short telomeres and improved organ fitness (Bernardes de Jesus et al., 2012). Furthermore, several studies also reported the role and importance of TERT for female reproductive tissues (Vasilopoulos et al., 2019, Butts et al., 2009, Treff et al., 2011. However, the effects of telomerase activation on ovarian tissues and female infertility have not been previously investigated. This study examined the effects of telomerase activation on ovarian tissues and its correlation with the anti-ageing Fgf21/Fgfr1/Klb signaling pathway. In addition to telomerase, the FGF21 pathway plays a critical role in ageingrelated metabolic disorders, such as insulin resistance, dyslipidemia, and obesity in rodents (Coskun et al., 2008, Kharitonenkov et al., 2005. A previous study reported that KL deficiency impaired telomerase activity in stem cells (Ullah et al., 2019). We found that, CAG-activated Tert upregulated J o u r n a l P r e -p r o o f the level of Klb in mouse primary GC culture. Prolonged culturing of mouse primary GCs, which induces ageing-like phenotypes, downregulated Klb protein level (Kulus et al., 2020). The downregulation of KLB upregulates the serum Fgf21 level and consequently promotes ageing (Villarroya et al., 2018). Treatment with FGF21 did not affect the downstream markers of FGFR1/Klb, such as Akt1 and mTOR in GCs cultured for a prolonged duration, which indicated that Klb downregulation suppresses FGF21 signal transduction (Villarroya et al., 2018, Fisher et al., 2010, Adams et al., 2012, Hu et al., 2022. Telomerase activation through CAG is an effective strategy to mitigate D-gal-induced ageing . We found that CAG can neutralize the D-gal induced ovarian toxicity by activating KLB/FGFR1 pathway via Telomerase activation. CAG mitigated the D-gal-induced downregulation of the KLB/FGFR1 complex and upregulated the protein levels of several critical proteins, such as Esr1 and Nr5a1 (Banerjee et al., 2012) (Liang et al., 2020). Furthermore, the GCs were treated with DOX to analyze the TERT inhibitory effects on the KLB/FGFR1 pathway (Eskiocak et al., 2008, Al-Kawlani et al., 2020a. DOX exerts adverse effects on GCs by inducing aromatase deficiency via downregulating estrogen levels (Al-Kawlani et al., 2020). However, we found that treatment with CAG significantly mitigated the DOX-induced downregulation of estrogen level.
We tried to analyze the potency of CAG against hTERT, but we did not find hTERT 3D structure in PDB. Therefore, we modeled our protein using template based and threading based approach with DS studio, Swiss-Model and I-TASSER webservers. The validated model was used for the study of molecular interactions study between CAG and hTERT. For this, molecular docking and molecular dynamics simulations were used. The molecular simulations revealed that CAG could bind with hTERT with strong binding affinity with -101.67 kJ/mol. The per residue contribution revealed that Arg901 contribute significantly in hydrophic interactions with hTERT. A recent modeling study revealed that the Arg631 and Tyr717 is important for the inhibitor binding with hTERT protein (Kalathiya et al., 2019). Interestingly, CAG was observed to interact with Arg631 through non-polar interaction. We therefore can conclude that interaction of CAG with hTERT in molecular docking and simulations, supporting the experimental results.
CAG is reported to exhibit various pharmacological activities, including TERT-activation, telomereelongation, antioxidant, and anti-inflammatory activities (Yu et al., 2018). Pharmacokinetic studies have revealed that CAG is absorbed by the intestinal epithelium through passive diffusion and undergoes first-pass hepatic metabolism (Zhu et al., 2010). Likewise, various experimental and clinical studies have indicated the safety of CAG, which has a wide range of applications (Yu et al., dysfunction, depression, and Osteoclastogenesis (Yu et al., 2018, Salvador et al., 2016. We investigated the effects of CAG on ovarian ageing and more specifically on GCs induced dysfunction due to ageing. It is well established by several studies that D-gal accelerate ovarian ageing by increasing oxidative stress in the ovary and enhances follicular atresia (Wang et al., 2019, Liang et al., 2020. We found that CAG significantly mitigated the D-gal induced damage to the ovarian follicles. Telomerase activation via CAG not only reduced the damage, but also enhances the key protein related to the GCs functioning (Nr5a1 and Foxl2). The malfunction of GCs, which results in age-associated reduced oocyte quality, is primarily characterized by altered telomerase activity (Iwata, 2017). We found that ageing not only reduced telomerase activity in the ovary but also alters the KLB signaling pathway.
Doxorubicin (DOX) is an effective antineoplastic drug, commonly used in childhood cancer. DOX is found to reduce the ovarian follicular size by inhibiting the oocyte surrounding granulosa cells (Aziz et al., 2020). Furthermore, DOX also altered the hormonal secretion and we found that DOX treated mice showed significant reduction in FGFR1/KLB pathway which is involved in female hormonal regulation. Interestingly co-treatment of CAG significantly mitigated the DOX induced ovarian follicular loss. DOX also induces mitochondrial apoptosis by disrupting the electron transport chain and compelling mitochondria to produce more ROS . It has previously been discovered that telomerase can link to mitochondrial DNA and protect it from damage (Haendeler et al., 2009, Singhapol et al., 2013. In order to understand CAG mediated telomerase activation effect on mitochondria, we found that CAG reduced the mitochondrial damage and also enhanced the NRF2 signaling which plays a noteworthy role in mitochondrial membrane potential (Holmström et al., 2016). Furthermore our results are in consistent with previous report which stated that telomerase-targeted therapy mitigates DOX-induced cytotoxicity (Chatterjee et al., 2021). We primarily attribute the healing of ovarian follicles to telomerase activation, which results in mitochondrial health, and subsequently to ovarian KLB protein level restoration.

CONCLUSIONS
This study elucidated novel functions of Telomerase in ovarian ageing. The findings of this study indicate that downregulation of TERT and Klb occurs in mouse ovarian ageing. The binding of CAG to the three-dimensional structure of hTERT revealed that CAG is an effective telomerase activator.
CAG-activated Telomerase not only upregulate Klb protein level but also improved mitochondrial functioning (non-nuclear function of Telomerase) to reduce the ovarian damage caused by D-gal or J o u r n a l P r e -p r o o f