FOSL2 regulates gonadotropin-dependent folliculogenesis through feedback amplification of FSH/FSHR signaling

25 Fosl2, a member of the AP-1 family, has been widely studied in the fields of 26 tumorigenesis and immune response, but its role in folliculogenesis remains unclear. 27 In this investigation, we presented comprehensive in vitro and in vivo evidence to 28 precisely define the biological functions of Fosl2 in folliculogenesis. Fosl2 in both 29 mouse and sheep, we demonstrated that the knockdown of Fosl2 effectively inhibited 30 cell proliferation and promoted cell apoptosis in both primary GCs and the GCs of 31 cultured gonadotropin (GTH)-dependent follicles. To explore the in-vivo function of 32 Fosl2, we generated an ovarian GC-specific conditional knockout (CKO) mouse 33 model. CKO mice showed impaired GTH-dependent folliculogenesis, leading to 34 disrupted estrous cycles and infertility in female mice. Subsequent bioinformatics 35 analysis and experimental results indicated that Fosl2 regulates the transcription of 36 FSHR and CYP11A1. These findings unveiled the essential role of Fosl2 in governing 37 the development of GTH-dependent folliculogenesis, thereby providing a novel 38 strategy for elucidating GTH-dependent folliculogenesis mechanisms and treating 39 ovarian dysfunction. 40

Fosl2, folliculogenesis, granulosa cell, estradiol, gonadotropin-dependent 41 follicle 42 43 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 3

44 Infertility has emerged as a significant global health concern, with impaired 45 folliculogenesis representing a critical factor in female reproductive dysfunction [1]. 46 Ovarian follicles, the fundamental functional units of ovary comprising oocytes and 47 granulosa cells (GCs), serve as pivotal determinants of female fertility. GCs orchestrate 48 folliculogenesis through multifaceted roles in oocyte -granulosa cell communication, 49 metabolic regulation, hormonal synthesis, and signal transduction [2-6]. The granulosa 50 cells are indispensable for oocyte maturation, GTH-dependent folliculogenesis (antrum 51 formation and expansion), and post-ovulatory luteinization [7-9]. The GTH-dependent 52 stage constitutes a decisive period governed by integrated endocrine networks 53 involving gonadotropins, steroid hormones, growth factors, and inhibins, which 54 collectively dictate follicular fate determination [10-13]. Elucidating regulatory 55 mechanisms during this critical developmental period remains essential for advancing 56 reproductive research. 57 Previous studies have reported numerous transcription factors that play key roles 58 in the ovaries [14-16].Fos-like antigen 2 (Fosl2), a component of the transcription factor 59 AP-1 family [17], modulates cellular processes including growth regulation, and 60 immune responses [18-26]. It has been reported that homozygous mice with systemic 61 knockout of Fosl2 died within one week after birth [27], and studies have also shown 62 that abnormal expression of Fosl2 can cause some diseases, such as asthma and 63 pulmonary fibrosis[28, 29], suggesting that Fosl2 may play an important role in the 64 regulation of animal organism . Although Fos family members regulate ovulation -65 related genes [30], and bioinformatic analyses implicate Fosl2 in polycystic ovary 66 syndrome [31]. The specific reproductive functions of Fosl2 in folliculogenesis remain 67 unknown. 68 This study demonstrates that Fosl2 deficiency disrupts GTH-dependent 69 folliculogenesis and induces female infertility. Transcriptome analysis revealed Fosl2 70 is highly expressed in ovarian granulosa cells and is induced by gonadotropin . GC-71 specific knockdown and conditional knockout impaired granulosa cell proliferation, 72 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 4 suppressed GTH -dependent folliculogenesis, and disrupted estrous cycl e. 73 Mechanistically, Fosl2 directly regulates the transcription of FSHR and CYP11A1 to 74 exert its function . Our findings establish Fosl2 as a n essential transcription factor of 75 GTH-dependent folliculogenesis, providing new insights fo r improving the 76 reproductive performance of humans and animals. 77

78 1. Characterization of the Fosl2 79 In order to further explore the transcription factors that play an important role in 80 the development of follicles , w e performed transcriptome analysis in mice ovarian 81 granulosa cells from PMSG 0h to PMSG 24h [32]. The expression of the transcription 82 factor Fosl2 was found to be significantly induced by PMSG (Figure 1A, B). The tissue 83 expression profile of Fosl2 in mice was mapped by qRT -PCR, confirming that Fosl2 84 was highly expressed in the ovary (Figure 1C). The r esults of qRT -PCR further 85 confirmed that Fosl2 expression was significantly induced by PMSG (Figure 1D.). 86 Immunofluorescence staining confirmed that Fosl2 was mainly localized in GCs of 87 ovarian follicles both in mouse and sheep (Figure 1E, F). These findings suggest that 88 Fosl2 is mainly localized in ovarian GCs and induced by PMSG. 89 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 5 90 Figure 1. Fosl2 is highly expressed in ovarian GCs and induced by PMS G (A) 91 Heatmap of the up-regulated genes in mGCs after PMSG injection. (B) Transcriptome 92 analysis was used to identify the up -regulated and down -regulated genes of 93 transcription factor in ovarian GCs after PMSG injection. Three GCs samples derived 94 from six mice per group were used for RNA-seq. (C) Tissue expression profile of Fosl2 95 gene in mice, n=3. (D) Expression of Fosl2 gene in mouse ovarian GCs 0, 24, 48 hours 96 after PMSG injection, n=5. (E) Immunofluorescence staining showed the localization 97 of Fosl2 protein in follicles of mouse . Green is a positive stain for Fosl2 protein. 98 Nuclear staining was performed with DAPI. (F) Immunofluorescence staining showed 99 the localization of Fosl2 protein in follicles of sheep. Green is a positive stain for Fosl2 100 protein. Nuclear staining was performed with DAPI . Statistical significance were 101 determined using one-way ANOV A followed by Tukey’s post hoc test , values were 102 mean ± SD. Significant differences were denoted by **P<0.0 1, ***P<0.005. The 103 experiments were repeated independently two times, yielding consistent results. 104 105 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 6 2. Knockdown of Fosl2 inhibits proliferation and promotes apoptosis in GCs 106 To investigate the function of Fosl2 in ovarian GCs, mouse primary GCs were 107 isolated and transfected with siRNA targeting Fosl2, with knockdown efficiency 108 confirmed (Figure 2A, B). Flow cytometric analysis of cell cycle distribution revealed 109 that Fosl2-knockdown significantly increased the proportion of cells in G1 phase while 110 reducing populations in S and G2 phases (Figure 2 C), indicating G1 phase arrest and 111 impaired DNA synthesis. Real-time cell analysis (RTCA) showed delayed entry into 112 the rapid proliferation phase and reduced cell index peak in Fosl2-knockdown GCs 113 (Figure 2 D). EdU staining further demonstrated suppressed proliferation in Fosl2-114 knockdown cells (Figure 2E). 115 Then the apoptosis of Fosl2-knockdown GC s was detected. Flow cytometry 116 analysis showed that knockdown of Fosl2 resulted in a significant increase in the 117 proportion of annexin -V-positive cells, indicating an increase in GCs at an early 118 apoptotic stage (Figure 2F). Knockdown of Fosl2 significantly increased the expression 119 of the apoptosis marker protein Cleaved -Caspase3 (Figure 2 G). Further TUNEL 120 staining showed that the proportion of apoptotic cells was significantly increased 121 (Figure 2H). Sheep primary GCs were isolated and transfected with siRNA targeting 122 Fosl2 (Figure 2 I, J). Similarly, apoptosis of cells is enhanced (Figure 2 K, L). 123 Conversely, Fosl2 overexpression (Figure S1A) reduced the proportion of early 124 apoptotic cells (Figure S1B), confirming the regulatory role of Fosl2 in apoptosis. 125 These results demonstrate that Fosl2 knockdown inhibits proliferation and 126 promotes apoptosis in ovarian GCs. 127 128 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 7 129 Figure 2. Fosl2 knockdown on GCs inhibits cell proliferation and promotes 130 apoptosis. (A-I) Knockdown Fosl2 in GCs of mouse. (A)Schematic representation of 131 the knockdown or overexpress of Fosl2 in primary mouse GCs. (B) Efficiency analysis 132 of Fosl2 interference using qRT-PCR, n=3. The scrambled siRNA was used as si-nc in 133 this study. (C) Effect of Fosl2-knockdown on cell cycle, left: Representative images of 134 cell cycle by flow cytometry, right: Cell cycle distribution, n=5(si-nc), 6(si-Fosl2). (D) 135 Effect of Fosl2-knockdown on rapid cell proliferation . (E) EdU staining of Fosl2 -136 knockdown cells, with red dots representing newly divided cells , left: Representative 137 fluorescence images of EdU staining, right: Cell proliferation rate. (F) Flow cytometry 138

of apoptosis after Fosl2-knockdown, left: representative images of apoptosis 139 detected by flow cytometry, right: Proportion of Annexin -V positive cells , n=3. (G) 140 Western blot assay of apoptosis related protein contents of Fosl2-knockdown primary 141 GCs, left: Expression of apoptosis -related proteins , right : Mean g ray values , n=3. 142 Original blots can be viewed in Figure S4A. (H) TUNEL staining of Fosl2-knockdown 143 cells, green represents apoptosis-positive cells , left: Representative fluorescence 144 images of TUNEL staining, right: Cell apoptosis rate. (I-L) Knockdown Fosl2 in GCs 145 of sheep. (I) Schematic representation of the knockdown or overexpress of Fosl2 in 146 primary GCs. ( J) Efficiency analysis of Fosl2 interference using qRT-PCR, n=3. (K) 147 Flow cytometry results of apoptosis after Fosl2-knockdown, left: representative images 148 of apoptosis detected by flow cytometry, right: Proportion of Annexin-V positive cells, 149 n=3. (L) TUNEL staining of Fosl2-knockdown cells, g reen represents apoptosis-150 positive cells, left: Representative fluorescence images of TUNEL staining, right: Cell 151 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 8 apoptosis rate . Statistical significance was determined using two -tailed unpaired 152 Student's t test or chi-square test, values were mean ± SD. Significant differences were 153 denoted by *P<0.05, **P<0.01 ***P<0.005, ****P<0.001. The experiments were 154 repeated independently two times, yielding consistent results. 155 3. Knockdown of Fosl2 impairs gonadotropin-dependent folliculogenesis 156 To investigate the direct effects of Fosl2 on folliculogenesis, we employed a 157 previously established in vitro follicle culture system of mouse [33] and sheep. The 158 expression of Fosl2 was disrupted by lentivirus transfection of shRNA in follicles at 159 different developmental stages. 160 Firstly, Fosl2 was knocked down in the pre-antral (GTH-independent) follicles of 161 mouse (Figure 3A), and observed fluorescence at 48 h after transfection (Figure 3B). 162 After the completed transfection, the follicles were transferred to fresh maturation 163 medium and cultured for 48h. The samples were collected after the follicular cavity can 164 be clearly observed. The interference efficiency was verified by protein levels (Figure 165 3B). T here was no significant difference in follicle volume and cavity area index 166 (Figure 3C). EdU staining of antral follicle sections further demonstrated no significant 167 difference (Figure 3D). Together, these data suggest that interfering with Fosl2 in 168 secondary follicles has no significant effect on their development. 169 Subsequent investigations focused on small antral (GTH-dependent) follicles. The 170 antral follicles were transfected and collected (Figure 3E, F). Measurements revealed 171 significant reductions in follicle volume and cavity area index, indicating restraint of 172 antral cavity expansion in Fosl2-knockdown follicles (Figure 3G). EdU staining of 173 follicular sections demonstrated suppressed granulosa cell proliferation (Figure 3H). 174 qRT-PCR analysis further showed downregulation of proliferation-related genes 175 (PCNA, Ki67, Cyclin E1 ; Figure S2) . These findings conclusively demonstrate that 176 Fosl2-knockdown impairs GTH-dependent folliculogenesis by suppressing antral 177 expansion and granulosa cell proliferation. 178 Moving on to sheep, Fosl2 was knockdown in antral follicles (Fig 3I, J). Similarly, 179 the volume of Fosl2-knockdown follicles was significantly reduced (Fig 3K). 180 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 9 181 Figure 3. Effect of Fosl2 knockdown on folliculogenesis at different stages. (A-D) 182 Fosl2 was knocked down in the cultured GTH-independent follicles in vitro. (A) 183 Schematic representation of the knockdown of Fosl2 in cultured GTH-independent 184 follicles. (B) Efficiency analysis of Fosl2 interference, left: Green fluorescence 185 indicates successful transcription of interfering plasmids in follicles, right: Western blot 186 assay of protein contents of Fosl2, n=3. Original blots can be viewed in Figure S4B. 187 The scrambled s hRNA was used as Control in this study. (C) Changes in the GTH-188 independent follicle volume, n=19 and follicular antrum index, n=4. (D) EdU staining 189 of GTH-independent follicles, left: Representative fluorescence images of EdU staining, 190 right: Cell proliferation rate, n=4. (E-H) Fosl2 was knocked down in the cultured GTH-191 dependent follicles in vitro. (E) Schematic representation of the knockdown of Fosl2 in 192 cultured GTH-dependent follicles. (F) Efficiency analysis of Fosl2 interference, left: 193 Green fluorescence indicates successful transcription of interfering plasmids in follicles, 194 right: Western blot assay of protein contents of Fosl2, n=3. Original blots can be viewed 195 in Figure S4C. (G) Changes in the GTH -dependent follicle volume, n=42 follicles 196 (Con), 37 follicles (sh-Fosl2) and follicular antrum index , n= 5 follicles (Con), 6 197 follicles (sh-Fosl2). (H) EdU staining of GTH-dependent follicles, left: Representative 198 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 10 fluorescence images of EdU staining, right: Cell proliferation rate, n=7 follicles (Con), 199 6 follicles (sh-Fosl2). (I-K) Fosl2 was knocked down in the cultured GTH-dependent 200 follicles of sheep in vitro. (I) Schematic representation of the knockdown of Fosl2 in 201 cultured GTH-dependent follicles of sheep. (J) Green fluorescence indicates successful 202 transcription of interfering plasmids in follicles. (K) Changes in the GTH -dependent 203 follicle volume, n=4. Statistical significance was determined using two-tailed unpaired 204 Student's t test or chi-square test, values were mean ± SD. Significant differences were 205 denoted by*P<0.0 5, **P<0.01 ****P<0.001. The experiments were repeated 206 independently two times, yielding consistent results. 207 4. Conditional knockout of Fosl2 in GCs leads to arrested GTH-folliculogenesis in 208 female mice 209 In order to further explore the effect of Fosl2 on GTH-dependent folliculogenesis 210 at individual level, we constructed a GCs-specific Fosl2 knockout mouse model (CKO). 211 We crossed Fosl2flox/flox with FSHR-Cre mice to create a GCs-Cre; Fosl2fl/fl mouse 212 (Figure 4A). Immunofluorescence staining and WB were utilized to verify the knockout 213 efficiency (Figure 4B, C). 214 An obvious estrus cycle disorder can be observed in adult female CKO-Fosl2 mice 215 of three estrus cycles (Figure 4D). Subsequent fertility assessments demonstrated 216 significantly reduced mating rates in CKO mice compared to Fosl2flox/flox mice, with 217 only one successful mating and no pregnancy (Figure 4E), indicating Fosl2 conditional 218 knockout induced female infertility. The serum estradiol content of mice also decreased 219 (Figure 4F). Since the estrous cycle is closely related to folliculogenesis, we counted 220 the ovarian weight of CKO-Fosl2 mice and found that the ovarian weight decreased 221 significantly after the conditional knockout (Figure 4 G), suggesting that there may be 222 abnormal folliculogenesis. HE staining of the ovarian sections at PMSG 48 h revealed 223 a significant reduction in the number of preovulatory follicle s (Figure 4 H), which 224 indicated that the arrested folliculogenesis led to the infertility of the mice. The 225 molecular phenotypes of ovarian GCs in CKO mice were also examined (Figure S3). 226 These findings collectively demonstrate that Fosl2 knockout impedes GTH-dependent 227 folliculogenesis, ultimately leading to female infertility. 228 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 11 229 Figure 4. Fosl2 conditional knockout result in infertility in female mice. (A) 230 Schematic representation of the Fosl2 conditional knockout in GCs of mice. Exon 2-4 231 deletion via FSHR-Cre-mediated recombination in GCs within GC; Fosl2flox/flox 232 (CKO). (B) Immunostaining showed that the Fosl2 gene was successfully knocked 233 out in ovarian GCs of CKO mice, blue: DAPI, green: Fosl2, red: Foxl2. (C) Western 234 blot assay of protein contents of CKO-Fosl2 mice, n=3. Original blots can be viewed 235 in Figure S4D. (D) Representative plot of estrous cycles, n=10. (E) Breeding rate, 236 pregnancy rates, litter size of CKO-Fosl2 mice. (F) Effect of conditional knockout of 237 ovarian GCs on estradiol levels in serum, n=3 serum samples. (G) Morphological 238 analysis of ovary after 48 hours of PMSG treatment, left: Representative photographs, 239 right: Statistical graph, n=4 ovaries (Fosl2flox/flox), 6 ovaries (CKO-Fosl2). (H) H&E 240 staining of ovaries 48 h after PMSG treatment, left: Representative photographs, 241 right: Follicle number statistics, n=3 ovaries (Fosl2flox/flox), 6 ovaries (CKO-Fosl2). 242 Statistical significance was determined using two-tailed unpaired Student's t test, 243 values were mean ± SD. Significant differences were denoted by **P<0.01 244 ****P<0.001. The experiments were repeated independently two times, yielding 245 consistent results. 246 247 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 12 5. Fosl2 regulates FSHR and CYP11A1 transcription 248 To elucidate the mechanism of Fosl2 in GTH-dependent folliculogenesis, we used 249 JASPAR and FIMO databases to predict the potential binding sites of Fosl2 protein to 250 key genes in folliculogenesis. Analysis showed that Fosl2 may target genes that play a 251 core role in GTH-dependent folliculogenesis (Figure 5A). The EMSA result revealed 252 FOSL2 binding to the Fshr promoter and Cyp11a1 promoter (Figure 5B). 253 To validate the aforementioned hypothesis, we examined the expression levels of 254 FSHR and CYP11A1 in Fosl2-knockdown GCs using qRT -PCR. The results 255 demonstrated that Fosl2-knockdown significantly downregulated the mRNA 256 expression (Figure 5C). Further, qRT-PCR analysis of Fosl2-knockdown cultured 257 follicle model indicated the similar results (Figure 5 D). Consistent results were 258 observed in GCs of CKO mice (Figure 5E). 259 These results suggest that FOSL2 can bind to critical genes’ promoter such as Fshr 260 and Cyp11a1. 261 262 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 13 263 Figure 5. FOSL2 directly bind to the promoter of Fshr and Cyp11a1. (A) Prediction 264 of possible binding sites of Fosl2 and GTH-dependent folliculogenesis genes. (B) 265 EMSA demonstrated FOSL2 binding to Fshr and Cyp11a1 promoter sequences. (C) 266 Expression of genes of FSHR and CYP11A1 of GCs in Fosl2-knockdown primary GCs, 267 n=3. (D) Expression of genes of FSHR and CYP11A1 of GCs after Fosl2 knockdown 268 in cultured follicles, n=3. (E) Expression of genes of FSHR and CYP11A1 in GCs of 269 CKO mice, n=4. Statistical significance was determined using two -tailed unpaired 270 Student's t test, values were mean ± SD. Significant differences were denoted 271 by*P<0.05, **P<0.01, ***P<0.005, ****P<0.001. The experiments were repeated 272 independently two times, yielding consistent results. 273 274 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 14

275 As a transcription factor affecting a variety of cells, Fosl2 is highly expressed in 276 ovarian GCs, but its effect on folliculogenesis remains underexplored. In this study, we 277 investigated the effects of Fosl2 on GTH-dependent follicles in vitro and in vivo. Fosl2 278 knockdown leads to proliferation arrest and apoptosis of primary GCs and GTH-279 dependent follicles. Notably, we demonstrate that granulosa cell -specific Fosl2 280 knockout causes female infertility in mice. 281 In previous studies, Fosl2 has been shown to be expressed and function in a variety 282 of cells and tissues. Including mediating renal tubular epithelial cell transdifferentiation 283 in fibrosis [34], suppressing myogenic differentiation in porcine muscle stem cells[35], 284 driving senescence in hepatic progenitor-like cells [36], negatively regulating NK cell 285 development [37]. The function of GCs is closely related to folliculogenesis and 286 maturation. Our identification of Fosl2's ovarian enrichment and PMSG -induced 287 expression suggests its critical involvement in folliculogenesis (Figure 1) . Further 288 studies showed that Fosl2 knockout had no significant effect on the growth of GTH-289 independent follicles, but only affected the GTH-dependent follicles (Figure 3). This 290 suggests that knockdown of Fosl2 inhibits folliculogenesis by inhibiting granulosa cell 291 proliferation and GTH-dependent follicle antral expansion. This is also consistent with 292 the effect of Fosl2 on inhibiting proliferation in other cells. 293 Since Fosl2 is stably expressed during the early development of animals and total 294 deletion leads to lethality, the conditional knockout method can be adopted for the 295 research on Fosl2 in specific tissues. Smith et al. have constructed conditional knockout 296 rats of Fosl2 in the pineal gland [38], and Chen et al. have constructed conditional 297 knockout mice with hematopoietic system deficiency [39]. In this study, after 298 conditional knockout of Fosl2 in mouse ovarian GCs, the mice showed disorders of 299 estrus cycles and infertility. The mating rate of CKO mice was extremely low, and they 300 did not become pregnant even after successful mating. The morphological analysis of 301 the ovaries suggested that the knockout of Fosl2 was accompanied by a decrease in 302 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 15 preovulatory follicles, which was consistent with its inhibitory effect on the growth of 303 GTH-dependent follicles (Figure 4). 304 Bioinformatics analysis and experimental verification indicated that Fosl2 can 305 regulate the expression of genes related to estradiol synthesis (Figure 5). This finding 306 is consistent with the conclusion that members of the AP -1 family are involved in the 307 transcription regulation of steroid synthetase [40]. At the same time, the expression of 308 genes related to granulosa cell differentiation FSHR, also significantly decreased while 309 the expression of genes related to estradiol synthesis CYP11A1, significantly decreased. 310 The direct regulation of FSHR and CYP11A1 by Fosl2 suggests that this regulation has 311 an important influence on some signaling pathways downstream of folliculogenesis [41, 312 42]. 313 At present, this study still has limitations. For future studies, we will further 314 explore the signaling pathways that Fosl2 regulate when it plays the role of regulating 315 folliculogenesis, and explore the expression and role of Fosl2 in males. In addition, the 316 role of Fosl2 in the regulation of folliculogenesis in sheep remains to be further studied. 317 The materials and methods employed in this study will be described in detail in the final 318 submission version. 319 In conclusion, this study demonstrates that FOSL2 directly regulates FSHR and 320 CYP11A1 transcription as an essential transcription factor for gonadotropin-dependent 321 folliculogenesis. Moreover, conditional knockout of Fosl2 in GCs of follicles leads to 322 disrupted estrous cycles and infertility in female mice. These findings elucidate the 323 important role of Fosl2 in the folliculogenesis process and fertility in female mice. This 324 study supplements the regulatory mechanism of GTH-dependent folliculogenesis and 325 provides a new approach for improving folliculogenesis, as well as new insights for 326 enhancing the fertility of female animals and humans. 327 328 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 16

329 Animals 330 Kunming mice were purchased from the Center for Animal Testing of Huazhong 331 Agricultural University (Wuhan, China). Fosl2flox/flox C57BL/6J mice were purchased 332 from GemPharmatech Co., Ltd., and FSHR -Cre mice were donated by Prof. Su 333 (Shandong University, China). Mice were reared in an SPF laboratory animal house, at 334 a constant temperature of 22±2℃, being allowed to access food and water ad libitum 335 with 12h light -dark cycles . Sheep samples are collected from slaughterhouses. All 336 experiments and handling of animals were approved and guided by the Institutional 337 Animal Ethics Committee of Huazhong Agricultural University Committee. 338 Construction of Fosl2 ovarian granulosa cell conditional knockout mouse model: The 339 exon2-exon4 of the Fosl2-201 (ENSMUST00000031017.10) transcript was used as 340 the knockout region. Ex vivo transcription of sgRNA was carried out to construct the 341 donor vector. Cas9, sgRNA and the targeting vector were injected into the fertilized 342 eggs of C57BL/6J mice through microinjection technology. F0 generation positive 343 mice were obtained by transplantation of fertilized eggs and verified by PCR and 344 sequencing. The F0 positive mice were crossed with C57BL/6J mice to obtain stable 345 F1 generation flox heterozygous mice models. F1 generation mice were mated with 346 each other to obtain Fosl2flox/flox mice. Fosl2flox/flox mice were crossed with FSHR-Cre 347 mice to obtain the conditional knockout heterozygotic mice of Fosl2 in granulosa cells 348 (Fosl2+/-Cre). Fosl2+/-Cre mice were crossed with Fosl2flox/flox mice to obtain the 349 conditional knockout mice of Fosl2 in granulosa cells, Fosl2flox/flox Cre (CKO-Fosl2). 350 Superovulation 351 Mice were injected 5IU pregnant mare serum gonadotropin (PMSG) (Ningbo Sansheng 352 Biological Technology) to promote follicle growth. 48 hours after injection of PMSG , 353 5IU human chorionic gonadotropin (hCG) (Ningbo Sansheng Biological Technology) 354 was injected to promote ovulation. 355 Analysis of RNA-seq 356 The mouse transcriptome data were sequenced in -house, with granulosa cells isolated 357 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 17 from pre -ovulatory follicles at 0- and 24 -hours post PMSG injection. Specific 358 transcriptome sequencing method was described in previous research [32]. 359 Analysis of qRT-PCR 360 Total RNA from samples was extracted using TRIzol reagent (Takara, 9109, Japan) and 361 cDNA was obtained by reverse transcription using Evo M -MLV RT Kit (AGbio, 362 AG11728, China). qRT-PCR was performed by CFX384 Real-Time PCR System (Bio-363 Rad). Reaction system includes: SYBR Green (Biosharp, China), 2 μL complementary 364 DNA template, 250 nM of the forward and reverse primers for each, and ddH 2O was 365 supplemented to a total volume of 10 μL. The reaction protocol was conducted as 366 described: an initial denaturation step at 95 ℃ for 10 min, succeeded by 35 cycles 367 comprising denaturation at 95 ℃ for 10 s and annealing/ extension at 60°C for 30 s. 368 Using ACTB to normalize gene expression levels, and using comparative 2 −△△Ct 369

to determine relative RNA quantification. The primer sequences are provided 370 in Table S1. 371 Western Blot 372 Extract total protein with lysis buffer consist of RIPA (ComWin Biotech, China), 373 protease and phosphatase inhibitors (ComWin Biotech, China) and PMSF (Solarbio, 374 China). Protein content was measured using BCA Protein Assay Kit (Servicebio, China). 375 Protein bands were separated sufficiently by SDS polyacrylamide gel electrophoresis 376 (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane. After blocking 377 the band with 5% skim milk powder (Nestle) at room temperature, incubated the band 378 overnight at 4 ℃ with the primary antibodies. Primary antibodies listed as follows: 379 Fosl2 (1:1000, Abclonal), 𝛼-Tubulin (1:1000, Biodragon -immunotech), Caspase3 380 (1:1000, Cell Signaling Technology), Cleaved -Caspase3 (1:1000, Abclonal), Bax 381 (1:1000, Cell Signaling Technology). Next, rinsed the bands with TBST (Servicebio, 382 China) and incubated with the secondary antibody: goat anti-rabbit immunoglobulin G 383 (1:4000, Biodragon-immunotech, China), goat anti-mouse immunoglobulin G (1:4000, 384 Biodragon-immunotech, China) for 120 min at room temperature. After rinsing with 385 TBST, the bands were visualized with ECL chemiluminescent reagent kit (Servicebio, 386 China) and acquire photographs with the Chemiluminescence Imager (Image Quant 387 LAS 4000 mini, USA). The housekeeping protein α-tubulin was used for gray value 388 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 18 statistic normalize. 389 Immunofluorescence Staining 390 The collected ovaries were embedded in 4% paraformaldehyde (Servicebio, China) to 391 be immobilized. After paraffin embedding, the ovaries were sliced into sections of 5 392 μm. Following dewaxing and antigen retrieval, the samples were treated with 0.5% 393 Triton-X-100 in PBS for 15 min to facilitate permeabilization, followed by blocking 394 with 5% donkey serum for 30 min. Subsequently, the sections were incubated with the 395 FOSL2 (1:100, Abclonal)/FOXL2 (1:100, Abclonal) antibody at 4 ℃ overnight. After 396 rinsing, the sections were incubated with the goat anti -rabbit immunoglobulin G (1: 397 10000, Biodragon-immunotech, China) at 37 ℃ for 60 min. Cell nuclei were stained 398 with DAPI at room temperature for 5 min. N ext, the samples were rinsed and 399 subsequently sealed with an anti -fluorescence quencher. Images were taken using the 400 LSM800 confocal microscope system (Zeiss, Germany). 401 Culture of primary granulosa cells 402 Primary g ranulosa cells were collected after puncturing mouse or sheep ovarian 403 follicles, filtered and inoculated into culture dishes. Cells were cultured at 37 ℃ in a 404 5% CO2 incubator using 10% fetal bovine serum (FBS) (Serana, Germany), DMEM/ 405 F12 (Gibco, Carlsbad, CA, USA) with 1% penicillin-streptomycin (Servicebio, China). 406 Subculturing after 48 h for subsequent experiments. 407 Culture of follicles 408 Mouse: The follicles of different sizes were isolated from mouse ovaries using 33-gauge 409 microneedles (KONSFI, China) for the experiment of follicles at different stages. The 410 size of Small preantral follicles is 100 -120 μm, and the size of antral follicles is 180-411 200 μm. Isolated follicles were cultured in 96-well plates (BKMAM, China), covered 412 with mineral oil (Sigma, Germany), and in an incubator maintained at 37 ℃ and 5% 413 CO2. The main components of follicle maturation medium include ：α-MEM (Gibco, 414 USA), 5% FBS (Serana, Germany), 1% ITS (Macklin, China), 100 U/mL penicillin –415 streptomycin (Servicebio, China) and 10 mIU/mL FSH (NSHF, China). 416 Sheep: The follicles of different sizes were isolated from sheep ovaries using 417 ophthalmic scissors and 26-gauge microneedles (KONSFI, China) for the experiment 418 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 19 of follicles at different stages. Isolated follicles were cultured in 96-well plates 419 (BKMAM, China), covered with mineral oil (Sigma, Germany), and in an incubator 420 maintained at 3 8.5 ℃ and 5% CO 2. The main components of follicle maturation 421 medium include ：α-MEM (Gibco, USA), 10% FBS (Serana, Germany), 1% ITS 422 (Macklin, China), 100 U/mL penicillin –streptomycin (Servicebio, China) ,50 μg/mL 423 ascorbic acid, 2 mM hypoxanthine, 2 mM glutamine and 10 mIU/mL FSH (NSHF, 424 China). 425 RNA Interference and Overexpression 426 Using si-RNA to inhibit the expression of target genes in Cells: When reached about 427 50% confluence, cells were transfected with siRNA using jetPRIME transfection 428 reagent. 48h later, cell samples were collected or conducted further experiments. 429 Lentivirus-mediated RNA interference was used to inhibit the expression of target 430 genes in follicles. Briefly, PLKO.1 -EGFP-PURO plasmid (Genecreate, China) was 431 utilized to construct interference vectors. Small interfering RNA targeted Fosl2 432 sequence is 5’- ATCATTGACCGCTCCTTTAGGT-3’. Negative siRNA, pMD2.G and 433 pSPAX were purchased from Genecreate. Lentiviruses were produced in 293 T cells 434 (ATCC, USA) by co-transfecting 4.8 μg interference vector, 2.4 μg pMD2.G, and 3.6 435 μg pSPAX2. The viral supernatants were harvested after 48 h, centrifuged, and filtered 436 through 0.45 μm polyvinylidene fluoride membranes (Sigma, USA). Follicles with a 437 specific diameter (mouse: around 140 µm, sheep: around 300 µm) were selected for 438 GTH-independent follicle knockdown Fosl2, and follicles with a diameter of around 439 180 µm were selected for GTH -dependent follicle knockdown Fosl2 to ensure that 440 Fosl2 was knocked down before follicular antrum formation. Follicles were cultured in 441 medium with 10 μg/mL polybrene and 100 μL/mL viral supernatants (mouse: 48h, 442 sheep: 96h). The medium was replaced with normal maturation medium after the green 443 fluorescence of the follicles was observed. 444 Using plasmid to overexpression the target genes in Cells: The protein coding region of 445 mouse Fosl2 gene (CCDS: CCDS19190.1, length 981bp) was inserted into the BamHI 446 XhoI cloning site of pcDNA 3.1 plasmid to construct an overexpression vector. 447 Transfection was completed using jetPRIME transfection reagent. 448 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 20 Flow Cytometry 449 Cell cycle: Primary granulosa cells transfected 48 h be collected and resuspended with 450 500 μL cold 70% ethanol at 4 ℃ overnight. After rinsing with PBS, the cell samples 451 were incubated with Rnase/PI at room temperature for 60 min. Early apoptosis: Primary 452 granulosa cells transfected 48 h be collected and resuspended with Annexin V -FITC 453 binding buffer. Add Annexin V-FITC/PI and incubate at RT for 15 min. Cell cycle and 454 apoptotic cells were estimated in a flow cytometer (Beckman Coulter). 455 Real-time Cell Analysis 456 The cellular dynamic proliferation was monitored using the RTCA DP Instrument 457 (Roche, Switzerland) based on electronic impedance detection. Cells were seeded in E-458 plate16 at an adjusted density of 7×10⁴ cells/mL. Subsequently, 100 μL of cell 459 suspension was dispensed into each well of the E -plate16. Following a 30 -minute 460 incubation at room temperature to facilitate cell sedimentation, the E -plate16 was 461 transferred to the RTCA station for continuous impedance monitoring. The proportion 462 of impedance change was continuously recorded, which was expressed by cell index , 463 with data acquisition performed at 30 -minute intervals over a 160 -hour observation 464 period. Cellular transfection was implemented at the 14th hour post -monitoring 465 initiation. 466 EdU Staining 467 Use EdU assay kit (Ribo Bio, China) to measure cell proliferation in cells and follicles. 468 Primary granulosa cells: 100 μL 50 µmol/L EdU be supplemented into the interfered 469 cells, and the cells were immobilized after 2h incubation. Follicles: 50 μL 1 mg/kg EdU 470 be supplemented into the interfered follicles, after incubating 24 h, used OCT (Sakura, 471 USA) embed the follicles and frozen, and employed Cryostat (Leica, Germany) to 472 section the follicle into 5 μm slices. Cell slides and follicle sections were incubated 473 with 1 × Apollo staining solution for 30 min . The nuclei were stained with 1× 474 Hoechst33342 reaction solution for 30 min. After staining and rinsing, images were 475 collected using a fluorescence microscope (Olympus, Japan). 476 TUNEL Staining 477 Cellular sample preparation method: The primary granulosa cell slides with interfering 478 factors were fixed with 4% paraformaldehyde (Servicebio, China) for 15 min, then 479 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 21 rinsed with PBS. Subsequently, 0.2% TritonX-100 in PBS was added and incubated at 480 37°C for 10 min. After rinsing with PBS, 100 μL of TdT Equilibration Buffer was added 481 to each sample and incubated at 37°C for 10-30 min. The TdT Equilibration Buffer was 482 then removed and the labeling working solution was added. Incubation was carried out 483 at 37°C in the dark for 60 min. After rinsing, DAPI was added and incubated for 5 min. 484 The samples were then sealed with an anti-fluorescence quencher. 485 The preparation method for frozen sections of follicles was the same as that for EdU 486 staining. Frozen sections were fixed with 4% paraformaldehyde (Servicebio, China) for 487 30 min, rinsed with PBS, and then 5 μg/mL proteinase K working solution was added 488 to each sample and incubated at 37°C for 10 min. The subsequent steps were the same 489 as those for cell slide staining. After staining, images were captured using a 490 fluorescence microscope (Olympus, Japan). Normal nuclei are shown in blue and 491 apoptotic cells in green. Apoptosis rate was the proportion of apoptotic cells to total cell 492 number. 493 H&E Staining 494 Vaginal smear staining : The dried vaginal smears were dyed with hematoxylin and 495 eosin respectively for 60 s, then fully rinsed and dried, and observed under a microscope. 496 Ovary section staining: Ovarian tissue sections were deparaffinized and rehydrated and 497 stained with hematoxylin for 6min, eosin for 20s . Then dehydrated with various 498 concentrations of alcohol and removed with xylene. Stained sections were sealed with 499 neutral gum, and images were obtained using a microscope (Olympus, Japan) for 500 analysis. 501 Estrus cycle determination and mating 502 Vaginal smears of sexually mature mice aged 8 weeks were collected daily for H&E 503 staining and the stage of estrus was examined for 15 days (3 estrus cycles) . The mice 504 that were confirmed to be in estrus were mated with male mice at night, and the vaginal 505 plugs were checked the next morning to ensure whether the mating was successful. 506 Prediction of binding sites 507 JASPAR (https://jaspar.genereg.net/) and FIMO ( https://meme-suite.org/meme/tool508 -s/fimo) were utilized to predict the binding sites of Fosl2 protein and gonadot509 ropin, focusing on follicle -related genes. 510 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 22 Electrophoretic mobility shift assay (EMSA) 511 The FOSL2 Coding sequence was cloned into the pcDNA3.1 -3XFlag plasmid 512 (Addgene, China) for overexpression. Flag -tagged FOSL2 proteins were 513 immunoprecipitated using an anti-Flag antibody (Beyotime, P2271, China). The elution 514 of proteins from the antibody was carried out with elution buffer (0.1 M glycine, pH 515 2.7) and then neutralized using a neutralization buffer (1 M Tris, pH 8.5). Biotin-labeled 516 DNA probes obtained from Genecreate (China) were utilized for the DNA EMSA, 517 conducted with the Chemiluminescent EMSA Kit (Beyotime, GS009, China), 518 following the manufacturer's instructions. In brief, recombinant Flag -FOSL2 and 519 biotin-labeled DNA probes were incubated in binding buffer for 30 minutes at room 520 temperature before being separated on a 4% native polyacrylamide gel at 100 V in TBE 521 buffer (Beyotime, R0223, China). Subsequently, the DNA -protein complexes were 522 transferred onto Amersham Hybond -N+ membranes (Cytiva, RPN1510B, USA), 523 blotted with HRP-conjugated streptavidin, and visualized via autoradiography. 524 Hormone Determination 525 Estradiol in serum was detected by radioimmunoassay kit (the Bioengineering Institute 526 China). Sera were obtained by centrifuging whole blood at 3000 rpm for 10 min . 527 Detection kit was purchased from the Bioengineering Institute (Nanjing, China) and 528 commissioned the North Institute of Biological Technology (China) for testing. 529 Statistics Analysis 530 Statistical analyses were using GraphPad Prism 10.0 (GraphPad). Data were expressed 531 as the mean ± SD. Two-tailed unpaired Student’s t test and one-way analysis of variance 532 followed by Tukey ’s post hoc test were used to analyze the statistical significance 533 between two groups and among multiple groups, respectively. Chi -squared test was 534 used in the comparison between the percentages. The statistical significance was set at 535 P-value <0.05. 536 537 538 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 23 DATA AVAILABILITY 539 All data are available from the corresponding author upon reasonable request. 540 FUNDING 541 This research was supported by the Fundamental Research Funds for the Central 542 Universities (2662023DKPY001) and the National Natural Science Foundation of 543 China (31701301). 544 SUPPORTING INFORMATION 545 This article contains supporting information. 546

547 We are grateful to Prof. Yongqiang Su (Shandong University, China) and Prof. Dr. 548 Louis Dubeau (University of South California, USA) for providing the FSHR-Cre 549 mice. 550 AUTHORS’ CONTRIBUTION 551 C.H. conceived, designed, conducted the experiments, analyzed and interpreted the data; 552 H.S., C.C. and Z.R. anticipated in experiment design and conduction, data analysis, and 553 manuscript preparation; J.L., Z.W., X.W., Y.Z., W.K., B.T. and Y.L. assisted with 554 sample collection and experiments conduction; C.H., H.S., W.R. C.C. and Z.R. wrote 555 the manuscript; X.L. and W. R. improved the manuscript. C.H., X.L., W.R. supervised 556 and funded this project. All authors approved the final version. 557 DECLARATION OF INTERESTS 558 The authors declare that they have no conflicts of interest with the contents of this 559 article. 560 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2025. ; https://doi.org/10.1101/2025.03.11.642212doi: bioRxiv preprint 24

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