Abstract Background This study aimed to analyze differentially expressed genes in theca cells of polycystic ovary syndrome (PCOS) rats using transcriptomic sequencing. Bioinformatics analysis and PCR validation were performed to identify genes involved in follicular development regulation in PCOS. Methods Twenty 6-week-old female SD rats with regular estrous cycles were divided into two groups (PCOS and control, n = 10 each). The PCOS model was induced with a 1.0 mg·kg⁻¹ Letrozole solution. Theca cells were collected for transcriptomic sequencing, and differentially expressed genes were analyzed. Functional annotations and pathway enrichment were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Gene interaction network and hub gene analyses were conducted, followed by RT-qPCR validation. Results PCOS rats exhibited increased body weight and irregular estrous cycles. A total of 1,114 differentially expressed genes were identified, including 516 upregulated and 598 downregulated genes. Fifty hub genes were selected for further analysis. GO and KEGG pathway enrichment analysis revealed significant involvement of the MAPK and PI3K-Akt signaling pathways. PCR validation confirmed that Cyp17a1, Cyp11a1, S6k1, mTOR, Akt, Kit, and Tek were significantly upregulated in the PCOS group (P < 0.05). Conclusion Transcriptomic analysis identified key genes and pathways involved in follicular development dysregulation in PCOS rats, providing potential targets for further research. Keywords: Theca cell, Follicular development, PCOS, Transcriptomic analysis Introduction Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders, affecting approximately 8–13% of women [[44]3, [45]31]. The primary pathological features of PCOS include follicular dysplasia, hyperandrogenemia, and irregular ovulation, which contribute significantly to infertility [[46]32]. Despite numerous studies, the exact molecular mechanisms underlying PCOS remain unclear [[47]30], and the identification of effective diagnostic and therapeutic strategies remains an urgent challenge in the field of reproductive medicine. Theca cells, which form part of the ovarian follicular membrane, play a crucial role in follicular development. These cells secrete androstenedione, which is converted by granulosa cells into estradiol, a key hormone involved in follicular maturation [[48]9, [49]36]. Additionally, the theca cell layer is rich in blood vessels, providing essential nutrients for follicular growth and maintaining a reciprocal relationship with granulosa cells and oocytes [[50]16]. However, in PCOS, theca cells exhibit dysregulated steroidogenesis, often leading to excessive androgen production, which disrupts follicular development and contributes to ovarian dysfunction. To date, research has primarily focused on the interactions between granulosa cells and the oocyte, while the role of theca cells has been somewhat overlooked in our understanding of ovarian physiology and pathogenesis [[51]34]. While much has been learned about granulosa cell function and the endocrine alterations in PCOS, the role of theca cells has been less extensively studied [[52]15]. Dysfunctional theca cells may play a central role in the development of the characteristic ovarian morphology seen in PCOS, such as follicular arrest and the accumulation of antral follicles. Therefore, understanding the molecular mechanisms that drive the dysfunction of theca cells is critical for elucidating the pathogenesis of PCOS. In this study, we utilized transcriptomic sequencing to analyze differential gene expression in theca cells from PCOS rats compared to the control rats. This sequencing method enables a comprehensive, genome-wide analysis of gene expression, providing valuable insights into the molecular changes in theca cells that may contribute to the disorder. Using bioinformatics tools, we identified key hub genes and signaling pathways involved in follicular development, with particular emphasis on the MAPK and PI3K-Akt pathways. These pathways are known to regulate ovarian function and may be disrupted in PCOS. By identifying and validating key genes and pathways involved in theca cell function, this study aims to provide novel insights into the pathogenesis of PCOS. Ultimately, these findings could inform the development of targeted therapeutic approaches that restore normal follicular development and improve fertility outcomes in women with PCOS. Materials and methods Experimental animals and ethical approval The 6w female Sprague-Dawley (SD) rats, exhibiting regular estrous cycles, were procured from the Experimental Animal Science Department of Shanghai University of Traditional Chinese Medicine. All rats received compassionate care in a temperature-controlled environment with a 12-hour light-dark cycle, and were provided ad libitum access to water and food. The animal procedures conducted in this study were approved by the Ethics Committee on the Use of Experimental Animals in Teaching and Research at Shanghai University of Traditional Chinese Medicine (PZSHUTCM2302060005). Study procedure Twenty 6w female SD rats were randomly allocated into two groups: the control group and the PCOS group, each consisting of 10 rats. Rats in the control group received normal saline via gavage over the same duration. Rats in the PCOS group were administered letrozole solution at a dose of 1.0 mg/kg via gavage, once daily for a consecutive period of 28 days. Throughout the modeling period, starting from the 3rd week, vaginal smears were collected from each rat every morning until the end of the study. The rats were kept until the second day after the 28-day modeling period before being analyzed. Finally, three rats were randomly selected from each group for the collection of follicular membrane cells, which were then used for transcriptomic sequencing. Vaginal smears and estrous cycle determination Vaginal smears were collected daily between 9 a.m. and 11 a.m. starting from the 3rd week of the experiment, prior to sacrifice. The stage of the estrous cycle was determined by examining the vaginal smears under a light microscope (Zeiss, Germany). Samples predominantly containing nucleated epithelial cells were indicative of the proestrus stage, while those primarily consisting of cornified squamous epithelial cells indicated the estrus stage. The metestrus stage was characterized by comparable quantities of cornified squamous epithelial cells and leukocytes, whereas the diestrus stage was dominated by leukocytes. Hematoxylin-eosin(HE) staining The ovaries were fixed with 4% paraformaldehyde, dehydrated conventionally, embedded in paraffin, and sectioned at a thickness of 4 μm. After routine deparaffinization and hydration, the sections were stained with hematoxylin-eosin for 15 min, followed by differentiation in 1% hydrochloric acid alcohol and staining with 1% eosin solution. The sections were then dehydrated, cleared, and mounted with neutral gum for observation of histopathological changes under a microscope. The ovarian specimens on the right side were stored in a -80 °C freezer for future use. Ovarian tissues from each group were subjected to HE staining, and their tissue morphology was observed under a light microscope, with counts of primordial follicles, early antral follicles, atretic follicles, and corpora lutea. Detection of sex hormones in rats All serum samples were processed according to the manufacturer’s protocol for each ELISA kit: follicle-stimulating hormone (FSH) ( IFU FSH CE, batch 3888), luteinizing hormone (LH) (IFU LH CE, batch 3927), and total testosterone (IFU Free Testosterone CE, batch 3960) (Immunodiagnostic Systems, Boldon, UK). Experiments were performed in triplicate and repeated three times. Theca cell isolation and identification Rat ovaries were collected, and follicles were dissected from the ovaries using a syringe. The follicles were punctured with a 1 mL injection needle to release granulosa cells. The remaining ovarian tissue was washed with M199 medium and then digested in M5A medium containing 5 g/L collagenase, 0.26 g/L DNase, and 10 g/L BSA for 90 min. The digested cells were centrifuged at 250×g for 10 min, the supernatant was discarded, and the pellet was washed once with M5A medium before being resuspended in 0.5 mL of M5A medium. The cells were purified using Percoll density gradient centrifugation. The resuspended cells were layered onto a gradient solution, with 2 mL of 35% gradient liquid on the upper layer and 1 mL of 50% gradient liquid on the lower layer. Centrifugation was performed at 1300 rpm for 10 min, and cells from the 35% gradient layer were collected. The collected cells were washed with an equal volume of M5A medium, centrifuged at 1500 rpm for 8 min, and the pellet obtained after centrifugation was identified as the theca cells. Theca cells were collected and counted using the Trypan. Blue exclusion method, with a viability rate exceeding 99%. The cells were then plated and cultured. To confirm the identity of the isolated theca cells, immunofluorescence staining was performed using an anti-CYP17A1 antibody (PROTEINTECH, 14447-1-AP, Rabbit Polyclonal). Additionally, immunofluorescence staining for the FSH receptor (FSHr) was conducted using an anti-FSHR antibody (AFFINITY, AF5242, Rabbit Polyclonal) to verify the absence of FSH receptor expression in the theca cells. RNA extraction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer(Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversly transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina(NEB #7530,New England Biolabs, Ipswich, MA, USA).The purified double-stranded cDNA fragments were end repaired, A base added, and ligated to Illumina sequencing adapters.The ligation reaction was purified with the AMPure XP Beads(1.0X).And polymerase chain reaction (PCR) amplified.The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Bioinformatics analysis GO functional analysis [[53]1], KEGG pathway analysis [[54]21] and Reactome Enrichment analysis [[55]28] were employed to elucidate and interpret the differentially expressed transcripts. The search tool STRING was used to analyse protein-protein interactions (PPI). The Cytoscape software was used to analyse the STRING-based DEGs interaction network. The Cytoscape plug-in tool MCODE was used to analyse the PPI network module to obtain the DEGs network hotspot module. MCODE score > 4 and the number of nodes > 5 were selected. The Cytoscape plug-in tool cytoHubba was used to identify key targets and sub-networks of complex networks. The top 50 genes with high degrees and with MCODE score ≥ 10 were utilized as the cut-off criterion [[56]12]. Reverse transcription quantitative PCR (RT-qPCR) cDNA was synthesized from 1 µg of total RNA using the first cDNA synthesis kit (Beijing Tiangen, China). RT-qPCR analysis was conducted using super premix (SYBR-green) (Beijing Tiangen, China). The RT-qPCR experiments were performed on the ABI StepOnePlus real-time PCR system in Foster City, California, USA. Specific primers for transcriptional amplification are listed in Table [57]1. Each sample was prepared in triplicate. The relative expression level of transcripts was calculated using the 2-ΔΔCT method with GAPDH as the standard. Table 1. Primer sequences for gene expression analysis in rats Gene Forward Primer (5’ -> 3’) Reverse Primer (5’ -> 3’) Akt CTCATTCCAGACCCACGAC ACAGCCCGAAGTCCGTTA CcnD1 GAGGAGCAGAAGTGCGAAGA GGCGGATAGAGTTGTCAGTGTAG Cyp11a1 GCTGGAAGGTGTAGCTCAGG CACTGGTGTGGAACATCTGG Cyp17a1 GATGGATGCACAGGCTGAGGTTAG GGAGGACCGTAGGAGGCACTG Eif4b GCAGAGACTATGACCGAGGC GCCTGTAATCGTCCCGAGAG Erbb3 ATCCGCTGGAATCATGAGGG ACTGGTTGTCTGCATCTCCG Gapdh CAAGTTCAACGGCACAGTCAAGG ACATACTCAGCACCAGCATCACC Hsd3b1 ATATTGGAGGCCTGCGTCG CGGCCATCCTTTTGCTGTA Kit TGCATTTAAAGGTAACAGCAAAGA GTGGCCTCAACTACCTTCCC mTOR TGCCTTCACAGATACCCAGTAC AGGTAGACCTTAAACTCGGACC Nos2 TGGTGAGGGGACTGGACTTTT GGGAATAGCACCTGGGGTTT Nos3 CAACTGGAAAAAGGCAGCCC GAGGTGGATCTCTCCTGGGT Pdgfrb CCGGCTACCCTATCTGGGAC AGTTGTCCTGGTTGCGGAAA Pik3 GTTCAGGATAAGTTCCGTCTGG GATGGGTCAAATCCACTTTCAT Rps6kb1 AAATCCGATCGCCTCGAAGA CACTTGTTTCCATTGGGTATTCCAC Star GCCTGAGCAAAGCGGTGTC CTG GCGAACTCTATCTGGGTCTGT Tek GGCAAGATGGATAGGGCTCA GCAGGCGGTAACAGTCTCAT [58]Open in a new tab Statistical analysis One-way ANOVA or paired sample t-tests were used to analyze all experimental data, with statistical significance set at P < 0.05. Differentially expressed genes were identified based on a fold change of ≥ 2 (absolute value). Results PCOS model evaluation Significant differences in body weight between the PCOS and control groups of rats began to emerge two weeks after letrozole administration. At weeks 8 and 9, the body weight (Fig. [59]1A) and the ovarian weight (Fig. [60]1B) of the PCOS rats was significantly higher than that of the control group. Additionally, starting from the third week of modeling, vaginal smears were performed on both groups of rats, and the results indicated that the estrous cycles of the PCOS rats became irregular (Fig. [61]1G). There were no statistically significant differences in the FSH levels between the two groups, but the levels of LH, LH/FSH ratio, and testosterone levels were significantly higher in the PCOS group compared to the control group (Fig. [62]1C, D, E, F). Histological analysis of ovarian tissue through H&E staining revealed that, compared to the control group, the ovaries of the PCOS rats exhibited significant polycystic-like changes, along with thickened theca cells (Fig. [63]1H, I). These results are consistent with the characteristic features of polycystic ovary syndrome. Fig. 1. [64]Fig. 1 [65]Open in a new tab Characteristic of the PCOS rat model. (A) Body mass of the PCOS model group and the control group. Significant weight differences began to emerge two weeks post-letrozole administration. At weeks 8 and 9, the body weight of the PCOS group was significantly higher than that of the control group (n = 10 per group). (B) Ovarian weight in the PCOS model group and the control group. At weeks 8 and 9, the ovarian weight in the PCOS group was significantly higher than in the control group (n = 6 per group). (C) FSH levels in the PCOS and control groups. There were no significant differences between the two groups (n = 10 per group). (D) LH levels in the PCOS and control groups. LH levels were significantly elevated in the PCOS group compared to the control group (n = 10 per group). (E) LH/FSH ratio in the PCOS and control groups. The LH/FSH ratio was significantly higher in the PCOS group (n = 10 per group). (F) Testosterone levels in the PCOS and control groups. Testosterone levels were significantly higher in the PCOS group (n = 10 per group). (G) Estrous cycle stages of the PCOS and control groups. Starting from week 3, irregular estrous cycles were observed in the PCOS rats, while the control group showed regular cycles (n = 10 per group). (H, I) Histological analysis of ovarian tissue. H&E staining revealed significant polycystic-like changes in the ovaries of PCOS rats, with thickened theca cells, compared to the control group (×40 and ×100 magnification, n = 4 per group). * P < 0.05. ** P < 0.01. *** P < 0.001. PCOS, polycystic ovarian syndrome. CTR, control Identification of theca cells Theca cells were collected, and the expression of CYP17A1 and FSHr was assessed by immunofluorescence. The results showed that CYP17A1 expression was positive in the theca cells, while FSHr expression was negative (Fig. [66]2A, B). Additionally, qPCR analysis of key genes involved in the steroidogenesis pathway, including Cyp17a1, Cyp11a1, Hsd3b1, and Star, was conducted in the theca cells. The results indicated that Cyp17a1 and Cyp11a1 expression in the PCOS group was significantly higher than that in the control group (P < 0.05), whereas Hsd3b1 and Star expression were also higher in the PCOS group, but without statistical significance (Fig. [67]2C, D, E, F). Fig. 2. [68]Fig. 2 [69]Open in a new tab Identification of theca cells. (A) Immunofluorescent staining of CYP17A1 (green) and DAPI (blue) in the theca cells. (B) Immunofluorescent staining of FSH receptor (FSHr) (green) and DAPI (blue) in the theca cells. (C) Relative expression of the Cyp17a1 gene in the theca cells from the PCOS and control groups. Cyp17a1 expression was significantly higher in the PCOS group (P < 0.05). (D) Relative expression of the Cyp11a1 gene in the theca cells from the PCOS and control groups. Cyp11a1 expression was significantly higher in the PCOS group (P < 0.05). (E) Relative expression of the Hsd3b1 gene in the theca cells from the PCOS and control groups. There was no significant difference between the two groups. (F) Relative expression of the Star gene in the theca cells from the PCOS and control groups. Star expression was higher in the PCOS group, but without statistical significance. * P < 0.05. ** P < 0.01. PCOS, polycystic ovarian syndrome. CTR, control Differential transcriptomic sequencing and bioinformatics analysis Compared with the control group, there were 1114 differentially-expressed genes (|log2FC|≥> log2(2), FDR < 0.05), including 516 up-regulated genes and 598 down-regulated genes (Fig. [70]3A, B). GO analysis was performed to determine the molecular functions (MF), cellular components (CC) and biological processes (BP) of the genes, and to predict the potential functions. In the Bar charts of GO classification, the top 20 pathways with the smallest P values were selected. The top BP enriched were mainly cellular processe, single-organism process, biological regulation, regulation of biological process and metabolic process. Their MF were mainly involved in binding, catalytic activity, molecular transducer activity, signal transducer activity and transporter activity. With the CC results showing that these genes were mainly involved in cell, cell part, organelle, membrane and membrane part (Fig. [71]3C). The main enrichment in KEGG signaling pathways includes ECM-receptor interaction, Pathways in cancer, Focal adhesion, AGE-RAGE signaling pathway in diabetic complications, and Pertussis (Fig. [72]3D). In Reactome pathways, the primary enrichment is observed in Extracellular matrix organization, Integrin cell surface interactions, Laminin interactions, Degradation of the extracellular matrix, and Procollagen lysyl hydroxylases converting collagen lysines to 5-hydroxylysines (Fig. [73]3E). Fig. 3. [74]Fig. 3 [75]Open in a new tab Differentially expressed genes and functional enrichment analysis. (A) Volcano plot showing differentially expressed genes (DEGs) between the PCOS model group and the control group. A total of 1,114 DEGs were identified, with 516 up-regulated and 598 down-regulated genes. The cutoff for significance was|log2FC| ≥ log2(2) and FDR < 0.05. (B) Heatmap of the top differentially expressed genes (DEGs) between the PCOS model group and the control group. The heatmap shows the expression levels of the 1,114 DEGs, with hierarchical clustering of genes and samples. (C) GO analysis of DEGs. The top 20 enriched biological processes (BP), molecular functions (MF), and cellular components (CC) were displayed. (D) Top 20 KEGG signaling pathways enriched in DEGs. (E) Reactome pathway enrichment analysis of DEGs. PCOS, polycystic ovarian syndrome. CTR, control PPI network construction and hub genes screening The STRING database compiles data on known and predicted protein interactions, and the protein network diagram illustrates the comprehensive network among proteins, with each node representing a protein. The names of differentially expressed protein groups were inputted into the official STRING website, and following analysis, the protein interaction network diagram presented was generated, consisting of 810 nodes and 12,929 edges. The top 50 differentially expressed genes (DEGs) were ranked by their degree in the network, with genes that have higher degrees (more connections) appearing darker, indicating their greater importance in the network(Fig. [76]4A). Three significant modules were identified through MCODE analysis: Module 1, with an MCODE score of 16.632, is the most highly interconnected and significant(Fig. [77]4B); Module 2, with an MCODE score of 15.538, is slightly less significant but still contains important key genes(Fig. [78]4C); and Module 3, with an MCODE score of 13.167, is the least significant of the three, though it still includes several connected genes(Fig. [79]4D). Additionally, the KEGG enrichment pathways of the selected hub genes were analyzed to provide insights into their biological functions. The hub genes are primarily enriched in the MAPK signaling pathway, PI3K-Akt signaling pathway, and IL-17 signaling pathway. ( Fig. [80]4E)。. Fig. 4. [81]Fig. 4 [82]Open in a new tab PPI network construction and hub genes screening. (A) Top 50 DEGs ranked by degree in the network. The higher the degree (more connections), the darker the gene appears, indicating its importance in the network. (B) Module 1, with an MCODE score of 16.632, indicating it’s a highly significant module with many interconnected genes. (C) Module 2, with an MCODE score of 15.538, slightly less significant than Module 1 but still an important module with key genes. (D) Module 3, with an MCODE score of 13.167, the least significant of the three modules but still important with several connected genes. (E) The KEGG enrichment pathways of the selected hub genes RT-qPCR validation We further validated the key pathways and genes identified in our analysis. The results revealed that the expression levels of mTOR (Fig. [83]5B), Akt (Fig. [84]5C), Rps6kb1 (Fig. [85]5E), Tek (Fig. [86]5H) and Kit (Fig. [87]5K) in the theca cells of PCOS model rats were significantly higher than those in the control group (P < 0.05), suggesting that these genes may play a critical role in follicular development in PCOS. However, no significant differences were observed in the expression levels of Pi3k (Fig. [88]5A), Eif4b (Fig. [89]5D), Nos2 (Fig. [90]5F), Nos3 ( Fig. [91]5G), Erbb3 ( Fig. [92]5I) and Pdgfra( Fig. [93]5J). Additionally, we also validated the expression of the cell cycle regulator CcnD1 (Fig. [94]5L), and found that its expression was significantly elevated in the PCOS model rats compared to the control group. These findings not only confirm the results of our transcriptomic analysis but also provide important insights for further exploring the molecular mechanisms of PCOS and potential therapeutic targets. Fig. 5. [95]Fig. 5 [96]Open in a new tab RT-qPCR validation. mRNA levels of cell cycle factors Pi3k (A), mTOR (B), Akt (C), Eif4b (D), Rps6kb1 (E), Nos2 (F), Nos3 (G), Tek (H), Erbb3 (I), Pdgfra (G), Kit (K) and CcnD1 (L) detected by qRT-PCR with GAPDH as an internal reference. * P < 0.05. ** P < 0.01 Discussion PCOS is a common endocrine disorder, one of the key features of which is impaired follicular development [[97]38]. In this study, a PCOS rat model was induced using letrozole (an aromatase inhibitor), which inhibits the conversion of androgens to estrogens, leading to elevated androgen levels and subsequent polycystic changes in the follicles [[98]18, [99]37]. In this model, the rats exhibited weight gain, irregular estrous cycles, enlarged ovaries with increased mass, elevated testosterone levels, an imbalanced LH/FSH ratio, and polycystic changes in the ovaries. These phenomena are consistent with the typical clinical features observed in PCOS patients. Therefore, this model effectively reflects the pathological characteristics of PCOS and provides an experimental basis for subsequent molecular mechanism studies. In the Two-cell hypothesis of gonadotropin action, theca cells play a crucial role in follicular growth and atresia. In addition to maintaining the structure of the follicle, another important function of theca cells is the synthesis of androgens [[100]39]. The function of the theca cells is disrupted in PCOS, with excessive proliferation, steroidogenesis imbalance, overproduction of androgens, and abnormalities in steroid synthesis pathways all contributing to follicular development disorders. These disturbances ultimately lead to ovulatory dysfunction and the formation of polycystic ovaries. The dysfunction of theca cells plays a crucial role in the pathogenesis of PCOS. A deeper understanding of this process is essential for uncovering the pathological mechanisms of PCOS and developing effective treatment strategies. In this study, we first examined the expression of steroidogenesis-related genes Cyp17a1, Cyp11a1, Hsd3b1, and Star in the theca cells. The Cyp17a gene encodes 17-α-hydroxylase, which plays a crucial role in the key steps of converting pregnenolone to 17-hydroxy-pregnenolone and progesterone to 17-hydroxyprogesterone [[101]22]. Previous studies have shown that the expression of Cyp17a is associated with hyperandrogenemia, hyperinsulinemia, and infertility risk in patients with PCOS [[102]2, [103]6, [104]10, [105]19]. Wicken Heisser et al. compared the activity of the Cyp17a promoter in the theca cells of normal and PCOS patients’ ovaries. They found that, compared to the normal group, the basal transcription levels and cAMP-dependent Cyp17 gene transcription were significantly increased in the theca cells of the PCOS group [[106]35]. Cyp11a is a crucial rate-limiting enzyme in the biosynthesis of pregnenolone from cholesterol. Pusalkar et al. found that elevated testosterone levels were associated with the presence of six repeat alleles in the Cyp11a1 gene [[107]25]. The study by Diamanti-Kandarakis et al. showed that variants of the Cyp11a allele are associated with both PCOS and total testosterone levels in women with PCOS [[108]5]. In addition, another study reported that the expression of Cyp11a is increased in the theca cells of PCOS patients, and in the PCOS group, the half-life of Cyp11a is almost twice as long as that in the normal group [[109]26]. This may be one of the factors contributing to the follicular changes observed in polycystic ovaries. These findings suggest that Cyp17a1 and Cyp11a could serve as genetic biomarkers for women with PCOS. Star (steroidogenic acute regulatory protein) regulates the transport of steroid hormones from the outer mitochondrial membrane to the inner membrane, while the Hsd3b1 gene is primarily involved in the conversion of pregnenolone to progesterone. Although our results did not show any significant differences in the mRNA expression levels of Hsd3b1 and Star between the PCOS and normal groups, studies have demonstrated that Hsd3b1 and Star play important roles in the development and progression of PCOS [[110]7, [111]14, [112]20]. In conclusion, we believe that Cyp17a1, Cyp11a1, Hsd3b1, and Star are candidate genes related to follicular development and hyperandrogenism in the ovaries of PCOS rats. These findings provide a foundation for further research. Although the ovaries are the primary source of androgens, other endocrine tissues are equipped with steroidogenic enzymes to produce androgens. For example, adipose tissue has been shown to possess enzymatic capabilities for androgen production, including 17α-hydroxylase activity [[113]24]. In our experiment, the rats showed weight gain and displayed clear symptoms of obesity. Mannerås et al. also demonstrated that letrozole-treated rats exhibited an increase in the weight of their primary adipose tissue stores. Therefore, the role of adipose tissue in the letrozole-induced increase in androgen production cannot be excluded [[114]17]. Using transcriptome sequencing, we analyzed the differentially expressed genes in the theca cells of PCOS rats. By comparing with the control group, we identified a series of differentially expressed genes related to follicular development, which were further validated through bioinformatics analysis and PCR validation. In total, we identified 1,114 differentially expressed genes, with 516 upregulated and 598 downregulated. Through PPI network analysis, we constructed an interaction network containing 810 nodes and 12,929 edges. To enhance the reliability of our results, we used Cytoscape to score the genes within the network and selected the top 50 genes with the strongest interactions for in-depth analysis. Further modular analysis of the PPI network using the MCODE plugin revealed 43 hub genes. Through KEGG and GO analysis of these 43 hub genes, we found that they were significantly enriched in the MAPK and PI3K-Akt signaling pathways. The MAPK signaling pathway plays a crucial role in biological processes such as cell proliferation, differentiation, and apoptosis [[115]29], while the PI3K-Akt signaling pathway is widely involved in cell proliferation, survival, and metabolism [[116]33]. In this study, the enrichment of the MAPK and PI3K-Akt signaling pathways suggests that these pathways may play a key regulatory role in follicular development in PCOS rats. Previous studies have shown that the MAPK pathway and the PI3K/AKT/mTOR pathway are critical in follicular development, and their abnormal activation may lead to impaired follicular development [[117]4]. We further found that the PI3K-Akt pathway was significantly enriched in the theca cells of PCOS rats, with the expression of related genes such as Rps6kb1, mTOR, and Akt being upregulated in the PCOS group. Overactivation of the PI3K-Akt pathway may lead to follicular developmental disorders by promoting ovarian cell proliferation and inhibiting cell apoptosis, ultimately affecting ovarian function in PCOS. Previous studies have also found that the PI3K-Akt pathway plays a key role in the pathogenesis of PCOS, particularly in ovarian endocrinology and the regulation of follicular development^[15–17]. Regarding the Nos2 and Tek genes, we found that they were not significantly enriched in these two pathways. Previous studies have shown that the overexpression of Nos2 in granulosa cells is associated with inflammatory anovulation in PCOS [[118]27]. However, its role in the theca cells has not been thoroughly investigated. Although our study shows a trend of decreased Nos2 expression in PCOS, the difference was not statistically significant. Tek, a receptor expressed in endothelial cells, plays a crucial role in angiogenesis [[119]8, [120]11, [121]23]. We also observed an upregulation of Tek in the theca cells of PCOS rats. CcnD1 is an important gene involved in cell cycle regulation. It plays a key role in cell proliferation and cell cycle progression. As a cell cycle regulator, CcnD1 primarily facilitates the transition from the G1 phase to the S phase, thereby promoting cell proliferation [[122]13]. Our study shows that the expression of CcnD1 is higher in the theca cells of the PCOS group compared to the control group. The upregulation of CcnD1 expression may be one of the reasons for the excessive proliferation of theca cells, which in turn affects follicular development. Although this study revealed the potential roles of several differentially expressed genes and signaling pathways in the theca cells of PCOS rats, there are still some limitations. First, this study focused solely on the transcriptomic analysis of the theca cells. Future research could expand to other ovarian cell types, such as oocytes and granulosa cells, to provide a more comprehensive understanding of the molecular mechanisms of PCOS. Second, although we identified the key roles of the MAPK and PI3K-Akt pathways in PCOS, the specific molecular mechanisms and regulatory networks of these pathways still need to be further validated through additional experiments. Although genes such as Pi3k, Eif4b, Pdgfra, Nos3, Nos2, and Erbb3 showed differential expression in transcriptomic analysis and had potential important roles in the Hub gene analysis, they did not reach statistical significance in qPCR validation. This could be attributed to various factors, including experimental methods, statistical thresholds, sample variability, and post-transcriptional regulation of genes. This does not imply that these genes are not important in the pathogenesis of PCOS, but rather suggests that their roles may need to be explored further using other analytical methods. Future studies with more rigorous experiments are needed for validation. In conclusion, this study revealed the follicular development regulatory mechanisms associated with PCOS through transcriptomic analysis. The MAPK signaling pathway and PI3K-Akt signaling pathway play important roles in follicular development in PCOS rats, providing new insights into the molecular mechanisms of PCOS and laying the foundation for the development of future therapeutic strategies. Acknowledgements