UFL1 Relieves Cisplatin-Induced Premature Ovarian Failure by Reducing Endoplasmic Reticulum Stress in Granulosa Cells

7 Background: Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1), the ligase of the Ufmylation system, has recently 8 been reported to be involved in apoptosis and endoplasmic reticulum stress (ER stress) in a variety of diseases. 9 Premature ovarian failure (POF) is a gynecological disease that severely reduces the fertility of women, especially 10 in female cancer patients receiving chemotherapy drugs. Whether UFL1 is involved in the protection from 11 chemotherapy-induced POF and its mechanism remains unclear. 12 Methods: In this study, we examined the function of UFL1 in ovarian dysfunction and granular cells (GCs) 13 apoptosis induced by cisplatin through histological examination and cell viability analysis. We used western blot, 14 quantitative real-time PCR (qPCR) and immunofluorescence (IF) to detect the expression of UFL1 and the level 15 of ER stress specific makers. Enzyme-Linked Immunosorbent Assays were used to detect the level of Follicle- 16 Stimulating Hormone (FSH) and Estrogen (E2) in ovaries and GCs. In addition, we knocked down or 17 overexpressed UFL1 in ovaries or GCs through infected with lentiviral particle suspensions, respectively. 18 Results: Our data showed that the expression of UFL1 was reduced in POF model ovaries and was accompanied 19 by the occurrence of ER stress. In vitro, cisplatin induced a stressful increase of UFL1 expression in GCs, and 20 enhanced ER stress, which was aggravated by UFL1 increase of atretic follicles, and a decreased expression of AMH and FSHR. Conversely, overexpression of UFL1 reduced the damage of cisplatin to the ovary in vitro. 24 Conclusions: Our research proved that UFL1 regulates cisplatin-induced ER stress and apoptosis of GCs, and 25 participates in the protection from cisplatin-induced POF, providing a potential therapeutic target for clinical 26 prevention of chemotherapeutic drug-induced POF. 27 continuous ER stress can induce the apoptotic pathway(35). In this study, our data also confirms that cisplatin causes apoptosis in GCs via triggering severe ER stress. To verify whether UFL1 plays a protective role in ER stress, we knocked down and overexpressed UFL1 in GCs and ovaries respectively. The results showed that OE- UFL1 can alleviate cisplatin-induced ER stress and apoptosis, reduce atretic follicles and improve ovarian function. Inversely, knockdown of UFL1 aggravate the damage of cisplatin. In summary, our results indicate that UFL1 plays a protective effect on GCs survive, follicular number and against follicle atresia via alleviating ER stress and apoptosis.


Quantitative Real-Time PCR 127
Total mRNA was extracted from tissue or cell samples using Trizol reagent. The cDNA was obtained by 128 reverse transcription of mRNA according to the instructions of the PrimeScriptRT kit for subsequent testing. TB 129 Green Mix was used for real-time quantitative PCR. The reference gene is β-actin, the PCR primer sequences are 130 shown in Table 1, and are used only for PCR amplification of specific segments of the gene of interest. 131 Table 1. Sequences used for quantitative real-time PCR. The JC-1 apoptosis detection kit (KeyGen BioTECH, China) was used to detect mitochondrial membrane 145 potential. GCs were washed three times in sterile PBS, and JC-1 working solution was added to each well and 146 incubated for 30 min under 5% CO2 and 37 °C conditions. Hoechst 33342 (Solarbio B8040) was used to stain the 147 nuclei. After incubation, the FGSCs were washed with PBS three times and observed under a NIKON Eclipse 80i 148 fluorescence microscope. The green fluorescent channel image (FL1) and the red fluorescent channel image (FL2) 149 were analyzed by ImageJ software, and the ratio of FL2 to FL1 was calculated to reflect the mitochondrial 150 membrane potential. 151

Hormone Measurement with Enzyme-Linked Immunosorbent Assays 152
For serum sample collection, at the end of the experiment, a blood sample was collected from the eyeball 153 vein and centrifuged at 3000rpm for 15 minutes. For in vitro cultured ovarian tissue samples and granulosa cells, 154 ovarian tissue homogenate and GCs suspension homogenate were collected after the required experimental 155 treatment. FSH (E-EL-M0511c, Elabscience, Wuhan, China) or AMH (E-EL-M3015, Elabscience, Wuhan, China) 156 levels in the samples were measured using ELISA kits according to the manufacturer's instructions. 157

Statistical analysis 158
Statistical analysis was performed using GraphPad Prism 8 software, and one-way analysis of variance was 159 used to detect statistical differences between multiple sets of data. P values <0.05 was considered statistically 160 significant. All data are expressed as the mean ± standard error of at least three independent experiments 161

The Expression of UFL1 Decreases in POF Model Mouse Ovaries 163
Firstly, we identified the expression of UFL1 in ovaries at different stages of development. During mouse 164 development from 1D to 10M, the expression of UFL1 increased from 1D to 2M and tended to decrease at 10M in 165 protein and mRNA level ( Figure 1A and Figure S1A), indicating that the abundance of UFL1 is associated with 166 ovarian aging. We constructed mouse models of POF by intraperitoneal injection of cisplatin ( Figure S1B-F). The 167 expression of UFL1 protein was significantly decreased in the ovary of POF model mice ( Figure 1B), and IHC 168 analysis showed that the decrease of UFL1 protein mainly occurred in ovarian GCs ( Figure 1C). Therefore, we 169 isolated primary ovarian GCs ( Figure S1G, H) and determined the expression and localization of UFL1 by IF in 170 GCs ( Figure 1D). Next, we treated GCs with a gradient concentration of cisplatin, the results discovered that the 171 protein expression of UFL1 increased within a certain concentration range (<15μM), whereas showed a decrease 172 at 20μM ( Figure 1E). Meanwhile, the level of UFL1 protein was elevated at 12h and reduced at 24h when treated 173 with 20μM cisplatin ( Figure 1F, G). In short, the expression of UFL1 was weaken in POF ovaries, and 174 instantaneously upregulated in stress and finally descended in GCs under cisplatin treatment.  To observe the damage effect of GCs caused by cisplatin, CCK8 analysis was used to detect the activity of 183 GCs. The results showed that the cell proliferation was inhibited by cisplatin with time and gradient dependence 184 ( Figure 2A, B). We next determined the occurrence of ER stress in POF GCs and ovaries, respectively. When 185 treated with a gradient concentration of cisplatin, the protein expression of ER stress specific markers GRP78 and 186 XBP1s in GCs increased within 15μM and decreased at 20μM while CHOP was persistently increased ( Figure  187 2C). Then, we chose 20μM cisplatin to analyze the changes of ER stress markers in GCs with different time points, 188 and the data discovered that the level of GRP78 and XBP1s increased within 12h and weakened after 24h while 189 CHOP raised slightly within 12h and upregulated significantly after 24h ( Figure 2D, E). As shown in Figure 2F

UFL1 Deficiency Aggravates Cisplatin-Induced ER Stress and Apoptosis in GCs 209
To confirm whether UFL1 is involved in the process of ER stress and apoptosis under cisplatin treatment, we 210 knocked down the expression of UFL1 with shRNA in GCs ( Figure 3A). Compared with the control group, GCs 211 proliferation was weaker in UFL1 knockdown groups ( Figure 3B, C), and the expression of the ER stress proteins 212 GRP78, XBP1s and CHOP were enhanced at the same time ( Figure 3D, E), which indicating that UFL1 depletion 213 were more vulnerable to ER stress and apoptosis. As shown in Figure 3F and 3G, the level of GRP78, XBP1s and 214 CHOP were significantly increased and a similar trend was observed both in the ratio of BAX/BCL-2 and Cleaved-215 Caspase 3/Caspase 3 in the UFL1 knockdown + cisplatin group. The IF images showed weaker red JC-1 signal 216 in the UFL1 knockdown +cisplatin groups than in the cisplatin treatment group ( Figure 3H), which suggesting that 217 UFL1 deficiency furtherly strengthened mitochondrial membrane potential (MMP) after cisplatin treated. In addition, 218 the alteration of E2 concentration also verified the above results ( Figure 3I). Together, our experiments proved 219 that UFL1 expression certainly correlates with ER stress and apoptosis induced by cisplatin.

Overexpression of UFL1 Resists Cisplatin Induced ER Stress and Apoptosis. 231
Further, we attempted to investigate the protective effect of UFL1 against cisplatin treatment via 232 overexpressed UFL1 (OE-UFL1) in GCs. As shown in Figure 4A, the level of UFL1 in the OE-UFL1 group was 233 more than two times compared with the control group. The results of CCK-8 analysis and EdU staining showed 234 that cellular viability and proliferation were increased after infected with UFL1 lentivirus particles ( Figure 4B, C), 235 and E2 level was increased simultaneously ( Figure 4D). Western blot results showed that overexpression of UFL1 236

The Loss of UFL1 Causes Ovarian Follicular Atresia 251
To evaluate the role of UFL1 in maintaining ovarian function, we co-cultured ovaries with UFL1 shRNA 252 lentivirus particles in vitro. As shown in Figure 5A, we successfully knocked down the expression of UFL1 in 253 ovaries. The FSHR and AMH represent ovarian reserve function, our results showed that their level were reduced 254 significantly after UFL1 knockdown ( Figure 5A), besides the concentration of E2 was also decreased in certain 255 extent ( Figure 5B). Compared with the control group, atretic follicles were added while primitive follicles were 256 decreased in the UFL1 knockdown group (Figure 5C-E). Same as the trend in UFL1 knockdown GCs, ER stress 257 markers also showed an obviously increasing in the ovarian UFL1 knockdown group ( Figure 5F). In addition, we 258 added UFL1 shRNA in cisplatin group. As a result, the expression ratios of apoptotic protein BAX/BCL-2 and 259 Cleaved-Caspase 3/Caspase 3 were obviously increased compared with only cisplatin group (Fig.5 G), and the 260 FSHR and AMH showed a uniform trend ( Figure 5H). Above all, these data indicate that the knockdown of UFL1 261 in ovaries induced follicular dysfunction, atresia and decline in number, suggesting that UFL1 might play a crucial 262 role in maintaining ovarian function.

3.6.UFL1 Alleviates POF Induced by Cisplatin in Vitro 273
Next, we co-cultured ovaries with OE-UFL1 lentivirus particles in cisplatin group, the efficiency of OE-UFL1 274 was shown in Figure 6A. After 7 days co-culture, the number of atretic follicles decreased and primordial follicles 275 increased compared with only cisplatin group ( Figure 6B-D), there was also an increased protein expression of 276 AMH and FSHR simultaneously ( Figure 6E). Furthermore, the protein level of BAX and Cleaved-Caspase3 were 277 decreased and BCL-2 was increased in the OE-UFL1 group ( Figure 6F), and that ELISA results showed an 278 increase of E2 concentration ( Figure 6G). Taken together, our data suggests that the overexpression of UFL1 can 279 mitigate POF induced by cisplatin and augment follicle number to some extent.

291
Recently researches have confirmed that UFL1 plays a crucial role in some biological process such as cell 292 proliferation, differentiation and embryonic development(25-28), but the function of UFL1 in POF has not been 293 explored. In this study, we firstly revealed that UFL1 is expressed in GCs, oocytes and stromal cells in ovarian 294 tissues. Comparing the expression of UFL1 in the ovaries at different developmental stages, it was found that the 295 level of UFL1 increased significantly along with follicular development, whereas decreased in aging ovaries. 296 Follicular development is accompanied with proliferation and differentiation of GCs, which implies plentiful protein 297 synthesis and posttranslational modification (PTM)(29, 30). Therefore, we speculated that the increased 298 expression of UFL1 contributes to the maintenance of ER homeostasis in GCs, and provides a stable internal 299 environment for follicular development. We detected the UFL1 protein level via constructing a POF model, the 300 results showed that UFL1 was decreased in POF mouse which supports our speculation. Next, we tested the ER 301 stress specifical molecules in POF model, and found that the level of GRP78 and XBP1s were decreased. 302 Interestingly, the expression of CHOP which can activate apoptotic pathway was increased obviously. Previous 303 studies have reported that ER stress can induce the expression of GRP78, XBP1s and other ER molecular 304 chaperones to produce protective effects as well as trigger endogenous cell apoptosis, and ultimately affect the 305 outcome such as adaptation, injury or apoptosis for stressed cells(31, 32). Therefore, we guessed that the 306 mechanism of POF caused by cisplatin is through severe ER stress, which eventually led to apoptosis of GCs, 307 follicular atresia and ovarian dysfunction. 308 ER stress is a transient and dynamic process, which molecular markers are highly susceptible to other 309 factors(33, 34). In order to avoid stimulation in ER homeostasis during the primary isolation and culture of GCs, 310 we treated normal GCs with cisplatin in vitro to replace primary POF GCs. The data showed that the level of UFL1 311 increased within a certain treatment time (<12h) in GCs with 20μM cisplatin. The interesting phenomenon was 312 that the changes of GRP78 and XBP1s showed a same trend as UFL1, while CHOP increased with the prolonged 313 treatment time which is consistent with alteration in POF ovaries. Cells occur the accumulation of a large number 314 of misfolded proteins after subjected cisplatin, and finally resulting in the occurrence of ER stress. showed that UFL1 firstly forms a receptor complex with C53 and DDRGK1, then DDRGK1 recruits UFL1 to the 329 ER surface for Ufmylation-dependent ER autophagy(39), and their binding is necessary for DDRGK1 Ufmylation 330 process(40, 41). And the C53 protein was an ER autophagy receptor, UFL1 and DDRGK1 are co-delivered to 331 vacuoles with C53 and are essential for C53-mediated autophagy(41). Therefore, the above researches suggest 332 that UFL1 changes the ER homeostasis by ER autophagy pathway. Beyond that, UFL1 may induce ER stress by 333 activating ferroptosis. As an important molecule in Ferroptosis, P53 can be covalently modified by UFL1 and 334 depletion of UFL1 can decrease P53 stability(42). Related study has shown that the activator of Ferroptosis 335 induces an increase in ER stress(43). Thus, UFL1 knockout may activate Ferroptosis and induce ER stress by 336 regulating P53 activity. In addition, the latest research has reported that the protein stability of SLC7A11, which is 337 a crucial ferroptosis regulator, can be reduced by inhibiting its Ufmyltion process(44). Thus, we speculate that 338 UFL1 deletion may can reduce the Ufmylation of SLC7A11 to activate ferroptosis related ER stress. 339 In this study, we focused on whether UFL1 can relieve the POF induced by cisplatin. The results indicated 340 that UFL1 alleviates ovarian dysfunction and GCs apoptosis by reducing ER stress in GCs, but we still need more 341 experiments in vivo to evaluate the potential function of UFL1 as a target to alleviate ovarian aging and prevent 342 POF caused by chemotherapy drugs. In conclusion, our study proved that UFL1 alleviated cisplatin-induced GCs 343 apoptosis and ER stress, and provide a new strategy and perspective to prevent the damage of chemotherapy 344 drugs in ovaries. 345