The Kazakh horse, a vital constituent of the equine family, plays an important role in human activities spanning production, daily life, competitive sports, and recreational pursuits. The distinctive seasonal patterns of estrus in Kazakh horses also impact their reproductive capabilities. Throughout the female reproductive cycle, follicles undergo continuous development, initially growing at a similar rate during each estrous period. Follicles, fundamental units of mammalian ovaries, require normal development to produce oocytes capable of fertilization (1). When a follicle, typically the largest, attains a critical size (approximately 35 millimeters in horses), a phenomenon known as diameter deviation occurs. This involves the continued growth of the dominant follicle (occasionally two or three follicles) while all other follicles (subordinate follicles) cease growth and enter a quiescent state (2). GCs, constituting a primary component of the follicle wall (3, 4), play a pivotal role in regulating follicular development. They not only provide nourishment to oocytes but also exert significant control over follicle growth and development through the secretion of hormones, cytokines, and protein regulation. Furthermore, they regulate the meiotic division process of oocytes and orchestrate follicular atresia through apoptosis. Post-maturation, follicular atresia is primarily induced by GCs (5). To discern the potential connection between granulosa cell proliferation and age (6), a growing body of research suggests that follicular atresia is mainly induced by apoptosis in GCs (7, 8). In horses, GCs stand out as the most crucial cell type within follicles, and their quiescence results in a diminishing reproductive function. Studies affirm that follicular atresia predominantly constitutes a hormone-regulated process involving GC apoptosis (9).

In females, oocytes remain in a state of meiotic arrest for several decades, with cycle lengths being similar (22 and 28 days, each featuring a 14-day luteal phase). Only one follicle ovulates per cycle, and the decline in oocyte quality has been identified as a major age-related factor in infertility (10, 11). Aging initiates a cascade of functional declines and diseases, leading to the cessation of reproductive function in both humans and animals. Given the substantial impact of aging on livestock production, the identification of molecular targets associated with aging holds crucial significance. Mares, due to their ovarian dynamics and reproductive capacity resembling humans, serve as crucial animal models for exploring ovarian aging (12). Research on reproductive lifespan remains an underexplored area. Follicles, as functional units of the reproductive lifespan, comprise oocytes (female gametes) and a specialized group of supporting cells. Unraveling the biology of follicles requires more dedicated efforts. In animal models, biological interventions aimed at manipulating pathways related to aging to maintain the quality and quantity of follicles may extend female reproductive and overall health span. Studies indicate intervention strategies targeting the molecular mechanisms driving ovarian aging and menopause (13). Therefore, investigating the mechanisms of follicular development plays a pivotal regulatory role in the reproductive and developmental processes of horses.

In recent years, with the advancements in biotechnology and functional genomics, whole transcriptomics has emerged as a crucial tool for delving into the characteristics and functions of biological cells. By scrutinizing the complete transcriptome of cells, distinctive gene expression patterns specific to each cell type can be discerned, thereby revealing information intricately linked to cell functions. Consequently, the exploration of the whole transcriptome characteristics and functions of ovarian GCs in Kazakh horses holds paramount significance for gaining a profound understanding of the cellular regulatory mechanisms governing the reproductive and developmental processes in this breed. Presently, there is a notable dearth of research in this specific domain. Thus, the primary objective of this study is to investigate the whole transcriptome of ovarian GCs in Kazakh horses, aiming to unravel their transcriptomic characteristics and the functions associated with them. This research endeavors not only to provide crucial support and guidance for the comprehensive study of reproduction and development in equine species but also holds the promise of offering novel insights and methodologies for genetic enhancement and the advancement of reproductive technologies in equine animals.

Ovaries and uterus, as essential reproductive organs, undergo the development and ovulation of dominant follicles on the ovaries, fertilization in the oviduct, and subsequent development in the uterus, leading to parturition under the influence of reproductive hormones. Transcriptomic studies related to ovaries and follicles have been applied to various species, such as humans (30), cows (17, 31, 32), pigs (33, 34), sheep (35), chickens (36), and geese (37). However, research on the ovaries of equine animals, especially horses, is relatively scarce. Horses, as typical seasonal breeders, experience estrus and ovulation influenced by various factors, including age, environment, and nutritional status. Reproductive aging in mares starts around 15 years old, and exceptional mares are essential for breeding high-quality racehorses, exemplified by horses like Dahlia, Allez France, and Oh So Sharp. Therefore, investigating the reproductive mechanism of mares using molecular biology techniques becomes crucial. The Kazakh horse exhibits evident seasonal estrus, occurring 2-3 times per year, with each estrus period lasting for 21 days. According to previous research by our team, mares aged 7-14 years in moderate-to-good body condition and exposed to a temperature range of 18-24°C show optimal estrus conditions. After 15 years of age, reproductive functions begin to noticeably decline, with a distinct pattern of follicular development and higher fertility compared to other age groups. This characteristic makes them an ideal animal model for studying mare follicular development. In-depth research has been conducted globally on the endocrine regulation of ovarian follicular development in equine animals, including mares. However, the specific molecules and cellular processes involved in the regulation of follicular growth and development at different ages in mares remain to be further explored.

In the comparison between groups A, B, C, and D in this study, 22 differentially expressed mRNAs exhibited significant downregulation, while FCER2 (ENSECAG00000009646) demonstrated a notable upregulation. Although existing research on FCER2 predominantly focuses on cancer, it is hypothesized that FCER2 plays a role in the regulation of ovarian aging. The varied expression states of these differentially expressed genes may regulate the proliferation and apoptosis of GCs, leading to follicular atresia or ovarian dysfunction. Among the 23 shared differentially expressed genes, AUTS2, ZFHX2, MAST4, SLC24A3, B3GNT6, and PPP1R13L showed a downregulation, while in Group A vs Group B, Group A vs Group C, and Group B vs Group C, they were upregulated. This suggests that AUTS2 is involved in regulating RNA transcription, splicing, localization, and stability. Follicular development and ovulation are highly complex biological processes, and lncRNA has emerged as a major translational regulatory factor in reproductive processes for humans and animals, such as gonadal development (38) and sex hormone secretion (39). GCs play a highly complex role in oocyte function, involving the regulation of multiple factors. This study suggests that lncRNA plays an important regulatory role in the follicular development and hormonal regulation of mares at different stages before sexual maturity, physical maturity, youth, and old age. By analyzing the lncRNA expression profiles, we identified differentially expressed genes in GCs at different ages in Kazakh horses. It is noteworthy that MSTRG.40650.1 showed significant upregulation, and its expression changed with age, suggesting that MSTRG.40650.1 may be involved in the regulation of aging. In Group C vs Group D, there were the most differentially expressed genes, with 74 upregulated and seven downregulated. MiRNA is a class of endogenous RNAs, approximately 18–24 nucleotides in length, which act by specifically binding to the 3’-UTR region of mammalian mRNAs to inhibit or reduce their translation, thereby post-transcriptionally regulating gene expression (40). Previous studies have shown that miRNAs play crucial roles in cell proliferation, differentiation, growth, migration, and apoptosis (41–44). MiRNAs may inhibit the expression of proteins involved in the proliferation and development of reproductive cells at the post-transcriptional level (45). Eca-miR-34b-3p, miR-12245-y, and miR-21-z show distinct expression patterns when compared under the same conditions, providing substantial evidence for their crucial regulatory roles in the sexual gland development of Kazakh horses. The CircRNA results indicate significant age-related features in the expression profile of GCs in mare ovarian follicle. Notably, novel_circ_001327 showed a downregulation in Group B vs Group C while displaying an upregulation in the other groups. Conversely, novel_circ_009685 exhibited upregulation in the B group compared to the C group but demonstrated downregulation in the other groups. novel_circ_008283 displayed a consistently decreasing trend across all stages. novel_circ_018383 and novel_circ_020541 exhibited an initial increase followed by a rapid decline, reaching their peak expression in the 7-year-old group (Group C) and then rapidly decreasing in mares aged 15 years and above (Group D). This suggests that novel_circ_001327, novel_circ_009685, novel_circ_018383, and novel_circ_020541 may be involved in signaling pathways related to proliferation, differentiation, and reproductive aging. Currently, there is limited research about the involvement of circRNAs, including novel_circ_001327, in ovarian and follicular development, and further studies are needed for validation.

Organisms achieve biological processes through the coordinated action of different genes and their products. The results of the GO enrichment analysis in this study show that differentially expressed genes in ovarian GCs of Kazakh mares at different ages are significantly enriched in biological processes and cellular components. The Mitogen-Activated Protein Kinase (MAPK) cascade is a highly conserved module involved in various cellular functions, including cell proliferation, differentiation, and migration (46). Mammals express at least four distinctly regulated MAPKs: Extracellular Signal-Regulated Kinase (ERK)-1/2, c-Jun N-terminal Kinase (JNK1/2/3), p38 proteins (p38 α/β/γ/δ), and ERK5. These MAPKs are activated by specific MAPKKs: MEK1/2 for ERK1/2, MKK3/6 for p38, MKK4/7 (JNKK1/2) for JNK, and MEK5 for ERK5 (47). However, each MAPKK can be activated by multiple MAPKKKs, increasing the complexity and diversity of MAPK signaling (48). It is speculated that each MAPKKK is responsive to different stimuli. For example, the activation of ERK1/2 by growth factors depends on the MAPKKK c-Raf, while other MAPKKKs may activate ERK1/2 under pro-inflammatory stimuli. The MAPK signaling pathway is one of the important pathways in eukaryotic signal transduction. MAPKs are activated by a series of extracellular stimuli, mediating signals from the cell membrane to the nucleus and regulating various physiological processes such as cell proliferation, differentiation, apoptosis, and death (49, 50). The Hippo signaling pathway is an evolutionarily conserved pathway that controls organ size from flies to humans. In humans and mice, this pathway consists of MST1 and MST2 kinases, their cofactors Salvador, and LATS1 and LATS2 (51, 52). When activated at high cell density, phosphorylated LATS1/2 activates the transcriptional co-activators YAP and TAZ, promoting their cytoplasmic localization, leading to apoptosis and restricting excessive organ growth (53). When the Hippo pathway is inactive at low cell density, YAP/TAZ translocates into the nucleus, binds to the transcription enhancer factor (TEAD/TEF) family of transcription factors, and promotes cell growth and proliferation (54). YAP/TAZ also interact with other transcription factors or signaling molecules, linking processes mediated by the Hippo pathway with other key signaling cascades, such as those mediated by TGF-β and Wnt growth factors (54). The Wnt signaling pathway is essential for the fundamental development of many different species and organs, regulating cell fate determination, progenitor cell proliferation, and controlling asymmetric cell division (55, 56). There are at least three different Wnt pathways: the canonical pathway, the Planar Cell Polarity (PCP) pathway, and the Wnt/Ca²⁺ pathway. In the canonical Wnt pathway, the primary role of Wnt ligands binding to their receptors is to stabilize cytoplasmic β-catenin by inhibiting the degradation complex, thereby stabilizing cytoplasmic β-catenin. Subsequently, β-catenin enters the nucleus freely, interacts with the T-cell factor (TCF) family of transcription factors and associated coactivators, and activates Wnt-regulated genes (57). This indicates that these differentially expressed mRNAs may participate in mare follicular development and the proliferation and apoptosis of GCs through the pathways mentioned above.

Mammalian TLRs are expressed on innate immune cells, such as macrophages and dendritic cells, and respond to membrane components of both Gram-positive and Gram-negative bacteria (58). TLRs recognize pathogens, inducing the production of pro-inflammatory cytokines and upregulating co-stimulatory molecules, thereby rapidly activating the innate immune response (59). The TLR signaling pathway is divided into two groups: the MyD88-dependent pathway, leading to the production of pro-inflammatory cytokines and the rapid activation of NF-Kappa B and MAPK, and the MyD88-independent pathway, inducing INF-β and INF-induced genes and slowly activating NF-Kappa B and MAPK. The MAPK signaling pathway is inhibited by prolactin and is crucial for normal follicular development (59, 60). Cell adhesion molecules are proteins on the cell surface that regulate physiological processes such as cell morphology, migration, proliferation, and differentiation (61). These findings suggest that lncRNAs play a crucial role in regulating protein-coding genes through both cis and trans actions, influencing follicular development in mares during estrus and follicular development. This, in turn, impacts the follicular development of mares at different ages.

Notably, differentially expressed miRNA target genes also exhibit enrichment in the Longevity regulating pathway - multiple species. GCs regulate the development and blockade of follicles through various factors, including gonadotropin receptors, steroid hormones, as well as various growth factors, and cytokines (62–64). Steroid hormones, including estrogen, progestogen, and androgen, directly regulate the maturation of follicles (65). Ras protein, as a GTPase and a molecular switch in signaling pathways, regulates cell proliferation, survival, growth, migration, differentiation, or cytoskeletal dynamics (66). It includes 627 genes, and the Ras signaling pathway is closely linked to 168 pathways, including the MAPK signaling pathway, Calcium signaling pathway, PI3K-Akt signaling pathway, and Regulation of actin cytoskeleton. The FOXO transcription factor family regulates gene expression in cellular physiological events, including apoptosis, cell cycle control, glucose metabolism, oxidative stress resistance, and lifespan (67). Besides PKB, JNK, and AMPK, FOXOs undergo various post-translational modifications, including phosphorylation, acetylation, methylation, and ubiquitination, involving 191,623 genes and 102 pathways, including the MAPK signaling pathway, Cell cycle, and PI3K-Akt signaling pathway. The PI3K-Akt signaling pathway, activated by various stimuli or toxic damage, regulates fundamental cellular functions such as transcription, translation, proliferation, growth, and survival (68). Once activated, Akt controls crucial cellular processes through the phosphorylation of substrates involved in apoptosis, protein synthesis, metabolism, and the cell cycle (69). Gonadotropin-releasing hormone (GnRH) is secreted by the hypothalamus, acts on receptors in the anterior pituitary, and regulates the production and release of gonadotropins, LH, and FSH (70). Downstream signaling of protein kinase C (PKC) leads to the deactivation of epidermal growth factor (EGF) receptors and the activation of MAPKs, including ERK, JNK, and p38 MAPK. Active MAPKs move into the cell nucleus, leading to the activation of transcription factors and the rapid induction of early genes (71). ECM-receptor interaction: The extracellular matrix (ECM) is a complex mixture of structural and functional macromolecules that play a crucial role in tissue and organ morphogenesis, as well as in maintaining the structure and function of cells and tissues (72). Specific interactions between cells and ECM are mediated by transmembrane molecules, primarily integrins, as well as possibly proteoglycans, CD36, or other cell surface-related components (73). These interactions directly or indirectly control cellular activities such as adhesion, migration, differentiation, proliferation, and apoptosis (74). Cellular senescence is an irreversible state of cell growth arrest triggered by various factors such as telomere shortening, oncogene activation, radiation, DNA damage, and oxidative stress (75). Its characteristics include enlarged and flattened morphology, increased activity of senescence-associated β-galactosidase (SA-β-gal), and the secretion of inflammatory cytokines, growth factors, and matrix metalloproteinases, forming part of the senescence-associated secretory phenotype (SASP) (76). Cellular senescence is functionally linked to various biological processes, including aging, tumor suppression, placental biology, and embryonic development. Ovarian Steroidogenesis is critical for normal uterine function, the establishment and maintenance of pregnancy, as well as mammary gland development. The ovarian steroids, 17-β estradiol (E2), and progesterone (P4) play pivotal roles in these processes (77).

This study established networks of protein interaction for differentially expressed genes in GCs of Kazakh horses across various ages, identifying core node genes within each group. Notably, TNF (Tumor Necrosis Factor) emerged as a core node in both Group B vs Group D and Group C vs Group D, exhibiting a significant upregulation in expression across both comparisons. TNF participates in 134 signaling pathways, including the MAPK signaling pathway, NF-kappa B signaling pathway, C-type lectin receptor signaling pathway, and TNF signaling pathway. Previous research indicates that TNF plays a dual role in follicular development across species like pigs and mice, promoting both cell survival and apoptosis. TNF stands out as a crucial cytokine capable of triggering diverse intracellular signaling pathways, encompassing apoptosis, cell survival, inflammation, and immunity. Activated TNF forms homotrimers and binds to its receptors (TNFR1, TNFR2), leading to the trimerization of TNFR1 or TNFR2. TNFR1, expressed in nearly all cells, serves as the primary receptor for TNF (also known as TNF-α). Conversely, TNFR2 is expressed in specific cell types, including CD4 and CD8 T lymphocytes, endothelial cells, microglia, oligodendrocytes, neuronal subtypes, cardiomyocytes, thymocytes, and human mesenchymal stem cells, acting as a receptor for both TNF and LTA (also known as TNF-β). Upon ligand binding, TNFR mediates the recruitment of adaptor proteins (such as TRADD or TRAF2), initiating signal transduction. TNFR1 signaling triggers the activation of numerous genes, predominantly regulated by two distinct pathways: the NF-kB pathway and the MAPK cascade, or cell apoptosis and necroptosis. TNFR2 signaling activates the NF-kappa B pathway, including the PI3K-dependent NF-kappa B pathway and the JNK pathway, promoting cell survival. TNF participates in GC apoptosis through the MAPK signaling pathway, and the apoptosis of GCs is directly linked to follicle development and the decline in ovarian function. The findings of this study suggest that TNF is similarly involved in regulating aging in large mammals beyond mice and humans (78), underscoring its high conservation. Additionally, TNF plays a critical role in multiple signaling pathways, including the mTOR signaling pathway, TGF-beta signaling pathway, and Cytokine-cytokine receptor interaction. Thus, the core nodes regulated by the protein interaction network present potential regulatory factors influencing the follicular development of Kazakh horses at different ages. However, a more in-depth investigation is required to elucidate the specific regulatory mechanisms. Another core node, KALRN, was identified in both Group B vs Group D and Group C vs Group D, exhibiting a significant downregulation in expression across both comparisons. This suggests that KALRN may regulate the proliferation and apoptosis of follicular GCs, thereby influencing follicular development through downregulation.

The ceRNA theory serves as a supplement to the traditional miRNA→RNA paradigm, shedding light on a reverse regulatory mechanism in RNA→miRNA interactions. In this context, ceRNAs competitively engage with the same miRNA recognition elements (MREs), thereby influencing the silencing of target genes triggered by miRNA (79). This theory unveils a novel mechanism of RNA interaction, garnering widespread attention in recent years across various domains, including tumor research, pathological studies, and investigations into growth and development. Currently, research on ceRNA mechanisms is predominantly focused on animals such as cows, pigs, sheep, chickens, and geese, with limited studies on horses. Particularly scarce are investigations into the regulatory network of ceRNA involved in regulating ovarian and follicular development. Through the analysis of mRNA differences in GCs of Kazakh horses at various ages, certain critical candidate genes related to follicular development have become the focal points of our study. Cell growth and differentiation, embryonic development, and the onset and progression of tumors are vital aspects of cellular physiology. The findings of this study reveal that within the ceRNA network, eca-miR-486-3p and miR-486-y have the highest number of nodes. Their target genes participate in multiple signaling pathways associated with reproduction, such as cGMP-PKG signaling, Basal transcription factors, Hippo signaling pathway, Longevity regulating pathway, Ovarian steroidogenesis, Endocrine and other factor-regulated calcium reabsorption, Cortisol synthesis and secretion, and more. Notably, ovarian steroids, 17-β estradiol (E2), and progesterone (P4) play a crucial role in normal uterine function, the establishment and maintenance of pregnancy, and mammary gland development (80). Furthermore, the locally essential actions for normal ovarian physiology rely on the endocrine, paracrine, and autocrine effects of E2, P4, and androgens (81). In most mammals, including humans and mice, ovarian steroidogenesis occurs based on the two-cell/two-gonadotropin theory. This theory elucidates how GCs and follicular cells collaborate to produce ovarian steroids (82, 83). Follicular membrane cells respond to LH signals by increasing the expression of enzymes needed to convert cholesterol into androgens (such as androstenedione and testosterone). GCs respond to FSH signals by increasing the expression of enzymes needed to convert androgens derived from the follicular membrane into estrogens (E2 and estradiol) (84). Cortisol is the primary endogenous glucocorticoid that influences various physiological functions, including lipid and glucose metabolism, metabolic homeostasis, and stress adaptation. Cortisol production is primarily regulated by adrenocorticotropic hormone (ACTH) from the pituitary gland (85). The stimulatory effect of ACTH on cortisol synthesis depends on cAMP-dependent signal transduction but also involves membrane depolarization and cytoplasmic Ca²⁺ increase. Both cAMP and Ca²⁺ induce the expression of StAR, stimulating cholesterol transfer within mitochondria the steroidogenic enzymes (such as CHE, CYP17A1, and CYP11B1) in the cholesterol-to-cortisol pathway (86, 87). While we have constructed a regulatory network related to the mechanisms of action in GCs at different ages and validated the accuracy of the data through RT-qPCR, the intricate mechanisms within the regulatory network still require further in-depth research and discussion.

This study employed whole transcriptome sequencing to analyze ovarian GCs from a total of 20 Kazakh horses across four different age groups. The results revealed that differentially expressed mRNAs and miRNA target genes were significantly enriched in pathways such as MAPK signaling, Hippo signaling, Wnt signaling, Calcium signaling, Aldosterone synthesis and secretion, Cellular senescence, and NF-kappa B signaling. By constructing protein interaction networks for mRNAs and miRNA target genes, potential regulatory genes associated with reproductive aging were identified. Noteworthy genes for further investigation in the context of horse reproductive aging and regulation include MCMDC2, TNF, and the core regulatory factors eca-miR-486-3p and miR-486-y. Functional validation at the cellular level will be conducted in the future to elucidate the specific mechanisms underlying differential gene expression in our experimental results.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal studies were approved by Laboratory Animal Care and Use Ethics Committee at Xinjiang Agricultural University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

WR: Conceptualization, Methodology, Writing – review & editing, Data curation, Investigation, Validation, Writing – original draft. JW: Investigation, Validation, Writing – original draft, Software. YZ: Visualization, Writing – review & editing. TW: Writing – review & editing, Formal analysis, Validation. JM: Project administration, Resources, Supervision, Writing – review & editing. XY: Writing – review & editing, Conceptualization, Funding acquisition, Methodology, Project administration, Supervision.