Theca Research Articles

The nomenclature for the female reproductive system was originally published in 2014. After 10 years of practical use, the scientific community requested from the organ working group (OWG) a review of the terminology and criteria for diagnosing ovarian sex cord/stromal lesions. As a result, OWG proposes the use of "sex cord/stromal" as the base terminology for hyperplasia and tumors to better reflect their origin from the sex cord/stroma and make the terminology internally consistent. When no predominant cell type is present, these lesions should then be designated as mixed cell type (e.g., "hyperplasia, sex cord/stromal, mixed," or "tumor, sex cord/stromal, mixed, benign"). When a clear, predominate cell type is present, the diagnosis should indicate that cell type (e.g., "sex cord/stromal, granulosa cell" or "sex cord/stromal, theca cell"). In the case of tumors, benign or malignant would be applied as appropriate. With these diagnostic revisions, the OWG for the female reproductive system attempts to provide clarification and refinement of criteria to be used for sex cord/stromal lesions.

FNDC4 modulates in vitro bovine granulosa and theca cell metabolism and alters follicle development in vivo.

Jiao-tai-wan and its component coptisine attenuate PCOS by regulating mitochondrial cholesterol import through suppression of SIRT1 ubiquitination.

The maturation of functional eggs in ovaries is essential for successful reproduction in mammals. Despite its biological and clinical importance, the underlying mechanisms regulating folliculogenesis remain enigmatic. Here, using murine ovaries, we report that the theca cells surrounding secondary follicles play a critical role in regulating follicle development through mechanical signalling. Using biophysical approaches, we found that the contractile theca cells exert significant compressive stress to the follicular interior through active assembly of fibronectin. Manipulation of compressive stress by targeting theca cell contractility, basement membrane integrity or intrafollicular pressure leads to changes in follicle size and mechanics, granulosa cell YAP signalling and oocyte-granulosa cell communications. Transcriptomics and quantitative immunofluorescence reveal that compressive stress impacts functional follicle growth through regulating the balance between granulosa cell proliferation and death that drives tissue pressure homeostasis. Altogether, our study uncovers unique mechanical functions of theca cells and provides quantitative evidence of the role of compressive stress in regulating mammalian folliculogenesis.

Disclosure: K. Vann: None. A. Weidner: None. A. Roy: None. O. Astapova: None.Polycystic ovary syndrome (PCOS) is a common and life-long endocrinopathy that affects around 10% of women and causes reproductive and metabolic derangements, culminating in infertility, increased cardiovascular risk and reduced quality of life. Its pathophysiology is driven by a vicious cycle of hypothalamic-pituitary-gonadal and metabolic hormone imbalance in which hyperandrogenism and insulin resistance potentiate each other through poorly understood mechanisms. To investigate the ovarian implications of systemic insulin resistance in PCOS, we utilized a chronic postnatal dihydrotestosterone (DHT)-exposed mouse model and verified that it develops both reproductive and metabolic manifestations of PCOS, including antral follicle arrest, cystic follicles and anovulation, excess adiposity, insulin resistance and compensatory hyperinsulinemia. Further, we isolated and cultured primary hepatocytes from these PCOS mice and confirmed that both insulin-induced hepatic AKT phosphorylation and FOXO1 nuclear export are impaired, as expected in insulin resistance. However, in primary ovarian granulosa and theca cells isolated from the same PCOS mice, there was no defect in these measures of insulin signaling, despite systemic insulin resistance, suggesting that the PCOS ovary is uniquely insulin-sensitive and exposed to chronic hyperinsulinemia due to systemic insulin resistance. To better understand the effect of hyperandrogenemia on ovarian insulin signaling, we treated primary mouse granulosa cells with DHT for 24 hours before stimulation with insulin. Both insulin-induced AKT phosphorylation and FOXO1 nuclear export were significantly reduced or absent after DHT exposure in this setting, indicating that hyperandrogenemia can initially impair insulin signaling in granulosa cells, but in PCOS the ovaries may overcome this defect over time. Thus, insulin stimulation of the PCOS ovary may be excessive due to chronic hyperinsulinemia and may contribute to ovarian dysfunction. In fact, PCOS theca cells demonstrated increased testosterone production when stimulated with both insulin and human chorionic gonadotropin. In summary, we show that the PCOS ovary is insulin-sensitive despite systemic insulin resistance, leading to excessive insulin stimulation due to chronic hyperinsulinemia, which may contribute to ovarian dysfunction in PCOS.Presentation: Saturday, July 12, 2025

Abstract Disclosure: A. Chen: None. Polycystic ovary syndrome (PCOS) is a multifactorial and common endocrine condition characterized by ovarian-source hyperandrogenism, abnormal folliculogenesis, insulin resistance, and infertility. While extensive knowledge through bulk transcriptomics has identified central molecular alterations in ovarian theca and granulosa cells, the cellular resolution of these phenomena at the single-cell level, most critically, those that underlie androgen excess and metabolic aberrations, is not yet adequately described. A high-resolution map of ovarian cell populations in PCOS is urgently needed to uncover the mechanisms of its heterogeneity and to inform the creation of targeted therapies. In the current study, we utilized scGPT, a cutting-edge machine-learning-based large language model, to combine and dissect multiple single-cell RNA sequencing (scRNA-seq) datasets from ovarian tissue of PCOS women and age-matched healthy controls. Leveraging its zero-shot generalizability, scGPT was able to identify sparse, rare, and transitional cell subpopulations in heterogeneous sample types without needing to be retrained, thus offering an adaptive and scalable cross-sample comparison platform. Compared with traditional methods such as scVI and Harmony, scGPT offered improved batch effect correction with preservation of key biological variability, particularly in the theca, granulosa, stromal, and immune compartments. Differential gene expression analysis revealed transcriptional programs corresponding to the hyperandrogenic and metabolically deranged phenotype of PCOS. Ovarian tissue from metformin-treated subjects, in particular, exhibited partial reversal of gene expression patterns observed in the disease, demonstrating the drug's potential to modify the ovarian microenvironment and restore molecular homeostasis. Our findings reveal the paradigm-shifting promise of machine learning-augmented single-cell genomics to untangle PCOS cell-type-specific disease mechanisms. In addition to gaining an unparalleled understanding of ovarian dysfunction, this approach also provides a critical foundation for precision diagnosis, treatment stratification, and future multi-omic integration in reproductive endocrine disease research. Presentation: Sunday, July 13, 2025

The earliest growing mouse follicles, wave 1, rapidly develop in the ovarian medulla, while the great majority, wave 2, are stored for later use as resting primordial follicles in the cortex. Wave 1 follicles are known to mostly undergo atresia, a fate sometimes associated with the persistence of steroidogenic theca cells, but this connection is poorly understood. We characterized wave 1 follicle biology using tissue clearing, lineage tracing, and scRNA-seq to clarify their contributions to offspring and steroidogenesis. Wave 1 follicles, lineage-marked by E16.5 Foxl2 expression in granulosa cells, reach preantral stages containing theca cell layers by 2 weeks. Atresia begins about a week later, during which 80–100% of wave 1 follicles degrade their oocytes, turn over most granulosa cells, but retain theca cells which expand in number together with interstitial gland cells in the medulla. During puberty (5 weeks), these cells ultrastructurally resemble steroidogenic cells and highly express androgen biosynthetic genes. Unexpectedly, the Foxl2 lineage tag also marked about 400 primordial follicles, located near the medullary–cortical boundary, that become the earliest activated wave 2 follicles. These ‘boundary’ or ‘wave 1.5’ follicles generate 70–100% of the earliest mature oocytes, while fewer than 26 wave 1 follicles with oocytes survive. Consistent with their largely distinct fates in steroid or oocyte production, granulosa cells of antral wave 1 and 2 follicles differentially express multiple genes, including Wnt4 and Igfbp5.

The earliest growing mouse follicles, wave 1, rapidly develop in the ovarian medulla, while the great majority, wave 2, are stored for later use as resting primordial follicles in the cortex. Wave 1 follicles are known to mostly undergo atresia, a fate sometimes associated with the persistence of steroidogenic theca cells, but this connection is poorly understood. We characterized wave 1 follicle biology using tissue clearing, lineage tracing, and scRNA-seq to clarify their contributions to offspring and steroidogenesis. Wave 1 follicles, lineage-marked by E16.5 Foxl2 expression in granulosa cells, reach preantral stages containing theca cell layers by 2 weeks. Atresia begins about a week later, during which 80–100% of wave 1 follicles degrade their oocytes, turn over most granulosa cells, but retain theca cells which expand in number together with interstitial gland cells in the medulla. During puberty (5 weeks), these cells ultrastructurally resemble steroidogenic cells and highly express androgen biosynthetic genes. Unexpectedly, the Foxl2 lineage tag also marked about 400 primordial follicles, located near the medullary–cortical boundary, that become the earliest activated wave 2 follicles. These ‘boundary’ or ‘wave 1.5’ follicles generate 70–100% of the earliest mature oocytes, while fewer than 26 wave 1 follicles with oocytes survive. Consistent with their largely distinct fates in steroid or oocyte production, granulosa cells of antral wave 1 and 2 follicles differentially express multiple genes, including Wnt4 and Igfbp5.

The earliest growing mouse follicles, wave 1, rapidly develop in the ovarian medulla, while the great majority, wave 2, are stored for later use as resting primordial follicles in the cortex. Wave 1 follicles are known to mostly undergo atresia, a fate sometimes associated with the persistence of steroidogenic theca cells, but this connection is poorly understood. We characterized wave 1 follicle biology using tissue clearing, lineage tracing, and scRNA-seq to clarify their contributions to offspring and steroidogenesis. Wave 1 follicles, lineage-marked by E16.5 Foxl2 expression in granulosa cells, reach preantral stages containing theca cell layers by 2 weeks. Atresia begins about a week later, during which 80-100% of wave 1 follicles degrade their oocytes, turn over most granulosa cells, but retain theca cells which expand in number together with interstitial gland cells in the medulla. During puberty (5 weeks), these cells ultrastructurally resemble steroidogenic cells and highly express androgen biosynthetic genes. Unexpectedly, the Foxl2 lineage tag also marked about 400 primordial follicles, located near the medullary-cortical boundary, that become the earliest activated wave 2 follicles. These 'boundary' or 'wave 1.5' follicles generate 70-100% of the earliest mature oocytes, while fewer than 26 wave 1 follicles with oocytes survive. Consistent with their largely distinct fates in steroid or oocyte production, granulosa cells of antral wave 1 and 2 follicles differentially express multiple genes, including Wnt4 and Igfbp5.

Fibrosis and tissue stiffening are hallmarks of ovarian ageing, linked to a decrease in fertility. However, the lack of three-dimensional (3D) characterization of ovary elasticity limits our understanding of localized elasticity patterns and their connection to tissue composition. Here, we developed an integrated approach to link ovarian elasticity, volume, and cell-matrix composition using quantitative micro-elastography (QME), a label-free, non-invasive method to study 3D microscale elasticity in conjunction with immunofluorescence microscopy. QME reveals distinct spatial elasticity patterns in ovarian compartments, namely follicles and corpora lutea (CLs), and local elasticity alterations in different age cohorts. CL elasticity significantly increases, and follicle elasticity changes minimally with age. CLs show size-dependent elasticity changes, while follicles exhibit distinct spatial variations in elasticity correlated with the emergence of theca cell layers during follicle development. These findings have the potential to guide novel diagnostics and therapeutic targets to improve women’s reproductive health and longevity.

Distinct gonadotropin receptor profiles across follicle sizes reveal potential mechanism for follicular co-dominance in goats.

Theca cells are a critical steroidogenic cell type of the ovarian follicle and corpus luteum. The ovulatory luteinizing hormone (LH) surge (or human chorionic gonadotropin (hCG)) stimulates theca cell relocation from the stroma surrounding the dominant follicle to full integration into the developing corpus luteum. LH/hCG also stimulates granulosa cells to produce local mediators of ovulation, including the peptide neurotensin (NTS). To determine if hCG-stimulated NTS regulates theca cell relocation within the ovulatory follicle, vehicle or a NTS receptor antagonist was injected into a macaque dominant follicle, and ovaries were removed 48 hours after hCG administration. Additional ovaries with dominant follicles were collected without administration of hCG (pre-hCG). Theca cells were sparse in the ovarian stroma surrounding pre-hCG follicles, while theca cells were abundant in the stroma and granulosa cell layer of recently-ovulated, hCG-treated follicles. Intrafollicular injection of a general NTS receptor antagonist or antagonist selective for a specific NTS receptor (NTSR1 or SORT1) reduced theca migration into the granulosa cell layer after hCG. In vitro, NTS stimulated macaque theca cell migration in conventional and 3-dimensional (3D) migration assays, and NTS receptor antagonists blocked NTS-stimulated migration. NTS-stimulated theca cell migration in vitro was influenced by ovarian extracellular matrix components, with laminin reducing theca cell migration. NTS also increased theca cell number in vivo and stimulated theca cell proliferation in vitro. In summary, hCG-stimulated NTS acts directly at theca cells via NTSR1 and SORT1 to stimulate theca cell migration during ovulation and transformation of the ovulatory follicle into the corpus luteum.

The yak is a large ruminant that lives in the high-altitude and hypoxic environment of the Qinghai-Tibet Plateau in China and typically exhibits limited reproductive capacity, posing a significant challenge to the advancement of animal husbandry in the region. Retinoid X receptors (RXRs), as an important member of the NR superfamily, play a key role in the regulation of reproductive hormone synthesis, follicular development, and embryo implantation. However, there is still a lack of systematic research on the expression characteristics and potential functions of RXRs in the yak's reproductive system. This study characterized RXR expression in ovarian, uterine, and oviductal tissues from three yaks per reproductive phase (follicular, luteal, and pregnancy). Using Quantitative Real-Time PCR Experiments (RT-qPCR), Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF), we analyzed RXR mRNA and protein expression and localization. RXR expression varied significantly (p ≤ 0.05), peaking in ovaries during the follicular phase, oviducts during the luteal phase, and uteri during pregnancy. RXRs were localized in ovarian granulosa and theca cells, oviductal epithelium, and uterine endometrial glands, with dynamic nuclear-cytoplasmic shifts. These findings suggest RXRs regulate key reproductive processes in yaks, offering insights on improving fertility in high-altitude environments.

Ovarian organogenesis requires the coordinated specification of supporting and steroidogenic cell lineages from multipotent coelomic epithelium (CE) progenitors. A longstanding question is whether the CE contains transcriptionally distinct, spatially organized progenitor subpopulations with predetermined lineage biases, or whether specification into supporting and steroidogenic lineages occurs only after delamination and integration into the bipotential gonad. The developmental origins of granulosa cells and the emergence of ovarian steroidogenic/stromal progenitors (SPs) also remain poorly defined. Here, we show that CE cells covering the fetal mouse ovary are transcriptionally heterogeneous and spatially organized into subdomains already primed toward supporting or steroidogenic fates. CE priming is dynamic, with transient coexistence of supporting- and steroidogenic-biased CE progenitors before resolving into a predominantly supporting-biased CE. Local delamination of these primed cells seeds intragonadal niches where pre-granulosa cells and SPs mirror the spatio-temporal arrangements of CE-primed progenitors. We further demonstrate a dual origin for the supporting lineage, with granulosa cells deriving from both the CE and supporting-like cells (SLCs). In parallel, we show that SPs arise from steroidogenic-primed CE cells, expand to represent 52% of ovarian somatic cells at birth, persist into adulthood and contribute to both theca and steroidogenic stromal cells. Together, these findings reveal transcriptionally and spatially distinct CE subpopulations that shape somatic lineage emergence with important implications for ovarian pathophysiology.

A reliable co-culture of granulosa (GCs) and theca cells (TCs) has the potential to facilitate a more comprehensive understanding of the interactions, signaling pathways, and substrate exchange between both cell compartments in the bovine ovarian follicle. Utilizing commercially available cell culture inserts, a reproducible co-culture model of bovine GCs and TCs was established. It was observed that primary theca and granulosa cells require considerably more time to attach to the insert membrane compared to cell lines. Initially, the TCs were seeded onto the inverted insert membrane, with a truncated tube serving as an inoculation container to prevent medium leakage. Following a 72 h incubation period, the insert was inverted, allowing the GCs to be cultured on the opposite side of the membrane. This co-culture was then subjected to an additional 6 days of incubation at 37 °C and 5% CO2, with media exchange every other day. The expression of marker genes indicated cell-type-specific patterns, with CYP17A1 being highly abundant in TCs and not expressed in GCs. Conversely, CYP19A1 showed high levels in GCs and only low levels in TCs. The hormone analysis supported the presence of a physiological co-culture system, as evidenced by the synthesis of estradiol. This modified co-culture model supports reproducible studies of paracrine signaling and substrate transport between the somatic cells of the follicle.

ABSTRACTThe causes of premature ovarian failure (POF) as a result of high‐fat and high‐sugar (HFHS) diets have not been studied systematically or in depth. In this study, we used single‐cell RNA‐seq (scRNA‐seq) and molecular pathology experimental techniques to systematically analyze the ovarian landscape in HFHS diet‐induced POF in mice. An HFHS diet decreased levels of AMH and E2 and induced a significant amount of follicular atresia in mice, according to the enzyme‐linked immunosorbent assay (ELISA) and pathology results. The scRNA‐seq results also showed that the number of Krt19 + epithelial cell, Csmd1 + cumulus cell, Mgarp + thecal cell, and mt‐CO1 + granulosa cell clusters was much higher in the ovarian tissues of HFHS–POF mice than it was in the control group. According to the KEGG analysis, the differentially transcribed genes in these three cell clusters in different groups were chiefly involved in multiple signal transduction pathways, and their overlapping signaling pathways included oxidative phosphorylation. Immunofluorescence staining and qPCR showed that expression levels of the oxidative phosphorylation signaling pathway were significantly lower in the control group than they were in the thecal cell, epithelial cell, and stromal cell clusters of the HFHS–POF mice. To the best of our knowledge, this study is the first to report the application of scRNA‐seq to analyze the ovarian landscape of HFHS‐fed mice and to verify that the HFHS diet induced POF in mice by activating the oxidative phosphorylation signal transduction pathway in the thecal cell, granulosa cell, and cumulus cell clusters in mouse ovarian tissues.

The endocrine system is crucial for maintaining overall homeostasis. However, its cellular signatures have not been elucidated during aging. Here, we conducted the first-ever single-cell transcriptomic profiles from eight endocrine organs in young and aged mice, revealing the activation of cell-type-specific aging pathways, such as loss of proteostasis, genomic instability and reactive oxygen species (ROS). Among six sex-shared endocrine organs, aging severely impaired gene expression networks in functional endocrine cells, accompanied by enhanced immune infiltration and unfolded protein response (UPR). Mechanism investigations showed that expanded aging-associated exhausted T cells activated MHC-I-UPR axis across functional endocrine cells by releasing GZMK. The inhibition of GZMK receptors by small chemical molecules counteracted the UPR and senescence, suggesting the immune infiltration is a possible driver of endocrine aging. Machine learning identified CD59 as a novel aging feature in sex-shared functional endocrine cells. For two sex-specific endocrine organs, both aged ovaries and testes showed enhanced immune responses. Meanwhile, cell-type-specific aging-associated transcriptional changes revealed an enhanced ROS mainly in aged theca cells of ovaries, while aged spermatogonia in testes showed impaired DNA repair. This study provides a comprehensive analysis of endocrine system aging at single-cell resolution, offering profound insights into mechanisms of endocrine aging.

Gonadotropins and anti-Müllerian hormone (AMH) regulate reproductive development and ovarian function. While AMH plays a well-established role in early folliculogenesis by counteracting gonadotropins, luteinizing hormone (LH) becomes more active later, during the antral phase, when granulosa and theca cells express their respective receptors. There are hints suggesting the existence of an interplay between these hormones regulating granulosa cell functions at late stages of the folliculogenesis., but the mechanisms remain unclear. In this context, we explored whether gonadotropin LH can modulate AMH action in cells of ovarian origin. Primary human granulosa lutein cells isolated from in vitro fertilization (IVF) patients were pretreated with recombinant LH, followed by stimulation with recombinant AMH. AMH receptor type II (AMHR2) expression, AMH signaling activation, and the expression of AMH-responsive genes were assessed through RT-PCR, ELISA, and Western blotting. The involvement of the LH receptor was confirmed using siRNA-mediated knockdown. Our findings showed that LH treatment downregulates AMHR2 transcripts and protein, impairing AMH-induced phosphorylation of small mothers against decapentaplegic (SMAD) 1, 5, and preventing osterix (OSX) and matrix metalloprotease 2 (MMP2) gene expression. These data are consistent with the possible interplay between gonadotropins and AMH in cells from antral/luteal stages and provide the molecular basis for further studies evaluating the impact of LH in modulating AMH signaling and follicular responsiveness during the antral stage.

Polycystic ovary syndrome: antimüllerian hormone and its role in the pathophysiology of the syndrome.

The primate ovarian reserve is established during late fetal development and consists of quiescent primordial follicles in the ovarian cortex each composed of granulosa cells surrounding an oocyte in dictate. As late stages of fetal development are not routinely accessible using human tissues, the current study exploits the evolutionary proximity of the rhesus macaque to investigate follicle formation in primates. Like in humans, the rhesus prenatal ovary develops multiple types of pre-granulosa cells in time and space, with primordial follicles deriving from later emerging pre-granulosa subtypes. In addition, our work shows that activated medullary follicles recruit fetal theca cells to establish a two-cell system for sex-steroid hormone production prior to birth, providing a cell-based explanation for mini puberty.