Comparison of follicle formation and molecular characteristics in reconstituted ovarioids under limited somatic cell conditions

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However, efficient production of reconstructed ovaries remains technically limited by the requirement for securing a large number of gonadal somatic cells. Therefore, this study evaluated the feasibility of follicle formation under limited somatic cell conditions by forming ovarioids with a reduced number of somatic cells. The formation of follicle-like structures was confirmed using immunofluorescence analysis, and the expression of molecular markers related to apoptosis, autophagy, mitochondrial stability, and stress responses was analyzed. As a result, it was observed that the groups with a reduced number of gonad somatic cells (2000:20000 and 3000:30000) formed follicle-like structures similar to the control group. Furthermore, oocytes observed under all conditions ranged in diameter from 50 to 70 µm, corresponding to the primary oocyte stage. Additionally, it was observed that the microenvironment was partially reproduced through staining of LAMININ, N-CADHERIN, and CD44. However, as the number of somatic cells decreased, differences were observed in the expression patterns of follicle-related and stress-related molecules. In particular, compared to the control group, the 2000:20000 group showed a significant decrease in follicle-related factors (Nobox, Lhx8, Gdf9, Bmp15). These results suggest that the number of somatic cells and the composition of the microenvironment in a reconstructed ovarian model can affect the qualitative environment for follicle formation, providing a basic basis for optimizing the conditions for ovarian reconstitution. Primordial germ cells Gonadal somatic cells Reconstituted ovary In vitro ovarian model Ovarian-like structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Oogenesis is the process by which primordial germ cells (PGCs) in the ovary develop from primary oocytes into mature oocytes through meiosis, mediated by interactions with gonadal somatic cells (GSCs) within the follicle (Wang et al. 2017; Nagaoka et al. 2021; Wei et al. 2024). During this process, GSCs support the growth of primordial germ cells (PGCs) through their interactions with them, and subsequently differentiate into theca cells and granulosa cells, contributing to the structural framework of the ovary (Zhang et al. 2014). Specifically, follicular somatic cells regulate meiotic arrest and progression, maintaining oocytes in a dormant state at the primordial follicle stage and inducing the resumption of meiosis in specific follicles in response to FSH (Umeno et al. 2022). Furthermore, follicular somatic cells induce follicular attrition and apoptosis during the follicular development cycle, ensuring that only the most viable follicles progress to ovulation (Wang et al. 2023). Therefore, the quality of the microenvironment formed by follicular somatic cells plays a key role in regulating ovarian structure, oocyte maturation, and overall reproductive success. Because follicles constitute a limited ovarian reserve at birth, early follicular formation and the stability of the microenvironment are crucial for maintaining female reproductive function (Zhang and Liu 2015). Based on germ cell-somatic cell interactions, in vitro oogenesis research in recent years has focused on preserving female fertility and enhancing our understanding of oogenesis mechanisms (Clarke 2018; Wang et al. 2017). Early research utilized oocyte-granulosa cell complexes isolated from newborn ovaries, which were cultured in two-dimensional (2D) in tissue culture well inserts to induce in vitro maturation (O'Brien et al. 2003; Jin et al. 2010). Subsequent development of three-dimensional (3D) culture systems has supported prefollicular formation while better mimicking the in vivo follicle structure (Khunmanee and Park 2022). These systems mimic the extracellular matrix and promote gap junction formation between primordial follicle cells (PGCs) and germline stem cells (GSCs), resulting in superior follicle formation compared to two-dimensional (2D) systems (Green and Shikanov 2016). Advances in in vitro oogenesis techniques have also included the cultivation of neonatal mouse ovaries. For example, fertile oocytes were produced by culturing female gonads isolated from embryonic day 12.5 (E12.5) mouse embryos (Eppig and O’Brien 1996; Morohaku et al. 2017). Subsequently, ovaries were formed by reaggregating primordial germ cell-like cells (PGCLCs) differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) with germ cell stem cells (GSCs) isolated from mouse E12.5. This is called a reconstituted ovary (r-ovary), and it is proposed as an experimental platform that enables follicle formation and oocyte development in vitro (Hayashi et al. 2012; Hikabe et al. 2016; Hayashi et al. 2017). However, oogenesis from mouse reconstituted ovaries remains limited by the difficulty in obtaining sufficient numbers of E12.5 GSCs, which provide the necessary environment for PGC growth (Wang et al. 2021). Typically, 5,000 PGCLCs and 50,000 GSCs are required to generate a reconstituted ovary. This requires the removal of residual PGCs and endothelial cells from approximately 2.5 female gonads, a labor-intensive and inefficient process (Hikabe et al. 2016; Hayashi et al. 2017; Sosa et al. 2023). This challenge is particularly pronounced in species such as humans, where fetal GSCs cannot be obtained (Hayashi et al. 2017). The differentiation process to replace this is expected to require approximately 75,000–100,000 GSCs (Yoshino et al. 2021). However, stable and scalable culture systems for GSC-derived cells remain limited. These limitations significantly impede the large-scale production of artificial ovaries. Nevertheless, complete elimination of GSCs from reconstituted ovaries is currently impossible because interactions between oocytes and somatic cells within the ovary are crucial for oocyte development (Lo et al. 2019; Vo and Kawamura 2021). Therefore, to address this issue, this study constructed structurally stable reconstituted ovarioids (r-ovarioids) with minimal GSCs and evaluated the feasibility of follicle formation under limited somatic cell conditions. The results demonstrated that follicle-like structures were observed in r-ovarioids with reduced somatic cell counts, partially reproducing the microenvironment derived from somatic cells. Conversely, significant differences in the expression of follicle- and stress-related molecules were observed depending on the number of somatic cells. These results demonstrate that follicle formation is possible even under limited somatic cell conditions and are expected to provide basic data for optimizing conditions for reconstructed ovarian models. Materials and methods All reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Animals All animal experiments were conducted in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Gyeongsang National University (Approval Number: GNU-240605-M0119). Eight-week-old CD-1 (ICR) mice obtained from Coretech (Seoul, Republic of Korea) were used for the experiments. The animals were maintained under a 12-h light/dark cycle with ad libitum access to food and water. Female mice were used on E12.5, based on the observation of vaginal plugs following natural mating with CD-1 (IGR) males. The day of vaginal plug detection was designated as E0.5. Gonad isolation An E12.5 pregnant mouse was euthanized by cervical dislocation, and embryos were carefully collected from the uterus. Embryos were washed several times with saline solutions to remove residual maternal tissues. Gonadal ridges were isolated from each embryo using a stereomicroscope. The gonads were separated from the surrounding mesonephros using a fine needle. Isolated gonads were dissociated into single-cell suspensions using 0.05% (w/v) trypsin-EDTA (Gibco BRL, Cat. No. 25300-062, USA) at 37°C. The dissociated cells were washed with a Leibovitz 15 medium (L-15; Cat. No. L5520) to neutralize trypsin and subsequently used for further experimental procedures. MACS MACS was performed according to the manufacturer's instructions (Miltenyi Biotec, Germany). Germline cells dissociated using 0.05% (w/v) trypsin-EDTA were resuspended in MACS buffer, which consisted of MACS® BSA Stock Solution (10% bovine serum albumin in phosphate-buffered saline (PBS), Cat. No. 130-091-376) and 0.5mM EDTA (Cat. No. E7889) in Dulbecco’s PBS (DPBS; Gibco BRL, Cat. No. 14190-250). The cell suspension was incubated on ice for 15 min with magnetic bead-conjugated antibodies, Anti-SSEA1 (Miltenyi, Cat. No. 130-094-530) and Anti-CD31(Miltenyi, Cat. No. 130-097-418). The mixture was then processed through MS columns (Magnetic Separation column) using a mini-MACS separation unit, following the manufacturer’s instructions. Non-magnetically retained cells were classified as PGCs, whereas magnetically retained cells were classified as GSCs. Cell counts were determined using a hemocytometer, and cell concentrations were adjusted as required for subsequent experiments. Reaggregation of PGCs and GSCs After adjusting the cell ratio to 1:10, the cells were reaggregated into three-dimensional cell aggregates under suspension culture conditions using 96-well U-shaped low-binding culture plates (Thermo Fisher Scientific, catalog no. 174925, USA). The cells were then cultured in a controlled humidity atmosphere of 5% CO 2 and 37.5°C for 3 days. To promote PGC expression, 0.3 µM retinoic acid (Cat. No. R2625) was added to the GK15 medium. The GK medium was prepared by mixing the following components: Glasgow's Modified Eagle Medium (GMEM; Gibco BRL, Cat. No. 11710035) with 15% (v/v) KnockOut™ Serum Replacement (Gibco BRL, Cat. No. 10828010), 1× MEM Non-Essential Amino Acids (Gibco BRL, Cat. No. 11140-050), 1 mM sodium pyruvate (Gibco BRL, Cat. No. 11360-070), 2 mM GlutaMAX™ supplements (Gibco BRL, Cat. No. 35050061), 1× penicillin-streptomycin (Cat. No. P4333), and 25.7 mM β-mercaptoethanol (Gibco BRL, Cat. No. 21985023). In vitro differentiation (IVDi) PGC and GSC differentiation was promoted using the IVDi culture method (Hayashi et al. 2017). Cultures were conducted in Transwell-COL membrane insert wells (Corning, Cat. No. 3493, NY, USA), to maintain the pre-established three-dimensional structure, with media changes every 2 days. Two types of culture media were used sequentially. MEM-based IVDi medium was used for the initial 4 days. This medium was prepared by mixing 2% (v/v) fetal bovine serum (FBS; Gibco BRL, Cat. No.16000-044), 0.15 nM ascorbic acid stock solution (TCI America, Cat. No. G0394), 2 mM GlutaMax, 1× penicillin-streptomycin, and 14.3 mM β-mercaptoethanol in MEM Alpha (Thermo Fisher Scientific, Cat. No. 12571-063). After four days, the cultures were maintained in StemPro-34-based IVDi medium. This medium was prepared by mixing, 10% (v/v) FBS, 0.15 nM ascorbic acid stock solution, 2 mM GlutaMax, 1×penicillin-streptomycin, and 14.3 mM β-mercaptoethanol in StemPro-34 SFM (Gibco BRL, Cat. No. 10639-011). The culture continued for 21 days. To prevent the formation of polycystic follicles, estrogen inhibitors were added from days 7 to 11 of the culture. The estrogen inhibitors were prepared by dissolving 12.14 mg of ICI 182,780 (Estrogen receptor antagonist, Tocris, Cat. No. 1047) in DMSO (Cat. No. D8418) to create a 20 mM stock solution. Isolation of secondary follicles After 21 days of culture, the r-Ovaries were dissected to isolate secondary follicles using a tungsten needle. Follicles in which the oocyte had ruptured, or the follicular structure had been damaged were excluded. After isolation, secondary follicles were evaluated in two ways: based on starting cell count (compared to PGC count) and based on constituent cells (compared to somatic cell count). JC-1 staining The mitochondrial membrane potential (Ψ) of oocytes was analyzed using JC-1 staining. After washing the live oocytes with PVA-PBS, 2.0 μg/mL of JC-1 was diluted in DPBS and incubated with the oocytes at 37.5°C in 5% CO 2 for 30 min. After incubation, the process should be performed in a darkroom, and the cells were washed with 2% (v/v) PBS-PVA and stained with DAPI for 5 min. Afterwards, it was washed again with 2% (v/v) PBS-PVA and observed using a laser scanning confocal microscope. The signal intensities of mitochondrial monomers (green) and aggregates (red) were measured using ImageJ. ROS analysis H2DCFDA was used to measure ROS levels. Oocytes isolated from secondary follicles and having their cumulus cells removed were placed in a PBS solution containing 1 nM H2DCFDA and incubated for 30 min at 37.5°C and 5% CO 2 . Afterwards, they were washed with PBS and photographed using an Olympus IX71 epifluorescence microscope. Extraction of complementary DNA (cDNA) and real-time PCR The r-Ovaries from IVDi culture on days 0 and 21 were washed three times with nuclease-free water, placed in a 1.5-mL tube containing 30 μL of nuclease-free water, and fresh-frozen in liquid nitrogen. Samples were stored at −80°C until processing. Total mRNA was extracted using a PicoPure RNA isolation kit (Thermo Fisher Scientific, Cat. No. KIT0204) according to the manufacturer's instructions. The concentration and purity of the mRNA were verified using a NanoDrop 2000/2000c spectrophotometer. The isolated mRNA was reverse transcribed into single-stranded cDNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Cat. No.1708891, Hercules, CA, USA). Gene-specific primers were designed using the Primer3Plus software (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) and are listed in Online Resource 1. The primers synthesized based on mRNA sequences were purchased from Macrogen (Seoul, Republic of Korea). Quantitative real-time PCR (qRT-PCR) was conducted on a CFX98 instrument (Bio-Rad Laboratories) using a 10-µL reaction mixture comprising 1× iQ SYBR Green Supermix (Bio-Rad Laboratories, Cat. No. 170-8882AP), 3 μL of diluted DNA, and 0.2 mM of each primer. All cDNA samples were normalized to the housekeeping gene, GAPDH , whose expression remained constant across all samples. Transcript quantification was performed using the ΔΔCt method, with normalized to the average GAPDH expression. The qRT-PCR thermal profile included an initial denaturation at 95°C for 3 min, followed by 44 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. The coefficients of intra- and interassay variance for each gene were calculated as follows: (standard deviation/mean) × 100. Immunofluorescence analysis and antibodies The r-Ovaries from IVDi cultures on day 21 were fixed with 4% paraformaldehyde at 4°C for 30 min and prepared for immunofluorescence staining. The r-Ovaries were washed three times with 0.2% (w/v) polyvinyl alcohol in PBS (PVA-PBS) for 10 min each. To enhance permeability, cells were treated with proteinase K for 5 min, followed by three washes. The samples were then incubated in a blocking solution (5% BSA in PVA-PBS) at 25°C for 90 min. After blocking, the r-Ovaries were incubated overnight at 4°C with primary antibodies (Online Resource 2). The samples were then washed three times with PVA-PBS and incubated with fluorescently labeled secondary antibodies (fluorescein isothiocyanate conjugated (FITC), tetramethylrhodamine isothiocyanate, and Alexa Fluor® 647; Santa Cruz Biotechnology, Dallas, Texas, USA) at 25°C for 90 min. For nuclear staining, the r-Ovaries were treated with DAPI (Thermo Fisher Scientific, Cat. No. 62248) at 25°C for 5 min. After the final wash, all r-Ovaries were mounted on glass slides using Anti-Fade Fluorescence Mounting Medium - Aqueous, Fluoroshield (Abcam, Cat. No. ab104135). Confocal imaging was performed using a laser scanning confocal microscope (Fluoview FV 1000, Olympus, Tokyo, Japan) and fluorescence intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij). Statistical analysis Statistical analyses of follicular development and gene expression levels between large and small groups were conducted using one-way ANOVA and t-tests in GraphPad Prism 6 (Graph Pad Software, San Diego, CA, USA), followed by Tukey’s post-hoc test for multiple pairwise comparisons between groups. Each experiment was performed in triplicate. Data are presented as means ± standard deviation, with significance defined at p < 0.05. Immunofluorescence and integral optical densities were quantified using ImageJ software and GraphPad Prism 6. Results Morphology and follicle of r-ovarioids with reduced somatic cell counts In this study, structures identified based on morphological characteristics in r-ovarioids were referred to as follicle-like structures, whereas follicles isolated and quantified were defined as follicles. In aggregates that were too small, the 3D structure collapsed when transferred to insert wells (Online Resource 3). From the 1000:10000 group, the 3D structure was maintained, but normal follicle-like structures were not observed after culture (Fig. 1a). On the other hand, in the 2000:20000 group, follicular morphology began to appear from day 14, and follicle-like structure was clearly observed on day 21 of culture. The 3000:30000 group also showed similar patterns, and the structure was maintained throughout the culture period, similar to the control group, 5000:50000. As expected, the control group observed the largest number of follicle-like structures (Fig. 1b), and the number of observed structures decreased as somatic cells decreased. After culture, structures resembling secondary follicles were observed in r-ovarioids, and morphologically maintained secondary follicles could be isolated from r-ovarioids. After directly isolating secondary follicles from r-ovarioids, the ratio of recovered secondary follicles was calculated based on the number of somatic cells (Fig. 1c). The 3000:30000 group showed a relatively high ratio, and the 2000:20000 group showed a similar level to the control group. In addition, when normalized based on the number of PGCs and analyzed, similar results were shown to the analysis based on the number of somatic cells (0.51 ± 0.10 vs. 0.71 ± 0.20 vs. 0.56 ± 0.25; p < 0.05; Table 1). Table 1 Comparison of r-ovarioid outcomes across PGC:GSC cell count ratios Cell ratios of PGCs: GSCs No. of r-ovarioids No. of total cells No. of PGCs No. of presumed secondary follicles (% ± SE) c No. of isolation secondary follicles (% ± SE) c 5000:50000 9 495000 45000 390 (0.87 ± 0.10) b 228 (0.51 ± 0.10) b 3000:30000 9 297000 27000 327 (1.21 ± 0.08) a 191 (0.71 ± 0.20) a 2000:20000 9 198000 18000 194 (1.08 ± 0.18) a 100 (0.56 ± 0.25) ab c Rates were calculated by normalizing the number of presumed or isolated secondary follicles to the number of PGCs. Three replicates were analyzed. p < 0.05 with different superscripts indicates the significant difference. r-ovarioids, reconstituted ovarioids; PGCs, primordial germ cells; 5000:50000, reconstructed ovarioids produced from a group of 5000:50000; 3000:30000, reconstructed ovarioids produced from a group of 3000:30000; 2000:20000, Reconstructed ovarioids produced from a group of 2000:20000. Presence of secondary follicles and oocytes in r-ovarioids Evaluation of the intrafollicular oocyte characteristics observed in r-ovarioids with reduced somatic cells revealed that all r-ovarioids expressed DDX4 (a germ cell marker), which was surrounded by NR5A1 (a germ cell somatic marker), suggesting the presence of follicle-like structures (Fig. 2a). DAPI staining revealed that the oocyte nuclei were at the germinal vesicle stage, showing an appearance consistent with the characteristics of secondary follicles (Fig. 2b). Furthermore, the diameters of oocytes isolated from secondary follicles ranged from 50 to 70 µm (62.73 ± 2.44 vs. 61.15 ± 1.78 vs. 59.89 ± 4.92; p < 0.05; Fig. 2c). Additionally, when we examined the mRNA expression of genes (Figla, Nobox, Lhx8, Gdf9, Bmp15) related to oocyte-specific transcriptional regulation and growth in r-ovarioid (Fig. 2d), a significant decrease in expression was observed only in Lhx8 and Bmp15 in the 3000:30000 group (p < 0.05, p < 0.01), whereas other genes did not show statistically significant differences. However, in the 2000:20000 group, except for Figla, the remaining genes showed significantly lower expression levels than the control group (p < 0.05, p < 0.001, p < 0.0001). Structural organization of somatic cells and expression of junction-related markers in r-ovarioids To evaluate the spatial characteristics of somatic cells and follicle-like structures in reduced r-ovarioids, LAMININ immunostaining was performed. Round follicle-like structures surrounded by LAMININ were observed in all r-ovarioids (Fig. 3a). The mRNA expression of early somatic differentiation-related factors (Foxl2, Wnt4) showed a similar trend to the control group in the 3000:30000 group (Fig. 3b). In contrast, the 2000:20000 group showed a significantly increased expression level compared to the control group, although the number of somatic cells was reduced (p < 0.05, p < 0.001). In addition, immunostaining for cell adhesion proteins (N-CADHERIN, CD 44) was performed to visualize the cell-cell contact patterns of somatic cells located around the follicle-like structures in r-ovarioids (Fig. 3c). As a result, N-CADHERIN signals were observed adjacent to the follicle-like structures, and ß-CATENIN, which stabilizes the interaction of N-CADHERIN, was also observed in all groups. Additionally, CD44 was also expressed on the surface of r-ovarioids in all groups. Analysis of the mRNA expression levels of cell junction-related genes (afadin, Cx43, Cx37) showed that only the expression of Cx43 was significantly increased in both the 3000:30000 group and the 2000:20000 group (p < 0.01, p < 0.001, respectively; Fig. 3e). Nr2f2, a GSC-derived stromal cell gene, was significantly increased only in 2000:20000 group compared to the control group (p < 0.05; Fig. 3f). Assessment of apoptosis- and autophagy-associated markers in r-ovarioids To evaluate cellular stress responses in r-ovarioid, the expression of apoptosis and autophagy-related markers was analyzed. Immunofluorescence signals of CASPASE-3 and BECN1 were observed within r-ovarioids in all experimental groups (Fig. 4a, b). Comparison of mRNA expression levels to determine the level of apoptosis (Bax, Bcl2, Perp) revealed that the 3000:30000 group exhibited similar expression levels to the control group (Fig. 4c). In contrast, the 2000:20000 group observed increased expression levels in Bcl2 and Perp (p < 0.05, p < 0.0001). These results demonstrate differences in the expression patterns of apoptosis-related genes under various conditions. Observation of functional stress-related changes in r-ovarioids To assess the functional stress state within r-ovarioid, the level of intracellular ROS production and mitochondrial membrane potential were analyzed. ROS levels significantly increased in the 2000:20000 group compared to the control group (Fig. 5a), and JC-1 staining results showed no differences across all groups (Fig. 5b). However, analysis of Tfam mRNA expression, an indicator of mitochondrial health, revealed decreased expression in the 2000:20000 and 3000:30000 groups compared to the control group (Fig. 5c). Furthermore, analysis of the expression of genes involved in DNA damage surveillance (Brca1, Chek2, Tap63) compared to the control group revealed that Brca1 and Chek2 increased in the 2000:20000 group, Chek2 decreased in the 3000:30000 group, and Tap63 showed no significant differences across all groups (Fig. 5d). In addition, comparative analysis of the Foxo3-Akt1 axis related to the regulation of follicular status showed that the expression of Foxo3 did not differ significantly in all groups, but the expression of Akt1 was significantly reduced in both groups compared to the control group (Fig. 5e). Discussion A limited ovarian pool of follicles and finite number of oocytes present substantial challenges in sustaining the reproductive potential (Chen et al.2014). To overcome this limitation, extensive studies have focused on oogenesis (Hayashi et al. 2017; Morohaku et al. 2017). Despite these advances, generating r-Ovaries requires a substantial number of GSCs, with approximately 1.5 times more GSCs required for applications after cryopreservation (Yoshino et al. 2021; Sosa et al. 2023). Even for differentiating large numbers of GSCs, non-mechanized protocols still pose challenges in meeting these requirements. However, because germ cells rapidly undergo apoptosis without support from GSCs, establishing efficient control of GSC numbers is a key area of need (Eppig 2001; Shen et al.2006; Dumesic et al. 2015). Therefore, this study compared r-ovaroids produced under conditions requiring a reduced GSC requirement to evaluate the characteristics of an in vitro culture system that can be utilized for oogenesis research. Research on ovarian reconstitution and follicular development has progressed in stages. Previous studies have reported that follicle-like structures can be obtained under conditions where 5,000 primordial germ cells (PGCs) and 50,000 germ stem cells (GSCs) are aggregated (Hikabe et al. 2016; Hayashi et al. 2017). Additionally, structures classified as primordial follicles are generally known to be surrounded by fewer than 10 granulosa cells in mice and 4–10 granulosa cells in cats and sheep (Lundy et al. 1999; Ariel et al. 2016; Wei et al. 2024). Therefore, in this study, r-ovarioids were cultured under conditions that allowed observation of follicle-like structures while maintaining the previously established cell ratio of 1:10. Initial attempts to form r-ovarioid with relatively small cell numbers (e.g., 1:10) resulted in difficulty in distinguishing cell aggregation, and limited observation of follicle-like structures in smaller groups (e.g., 10:100, 20:200, 50:500). This suggests that a certain number of cells may be required for r-ovarioid formation. In addition, according to previous studies, the composition of the follicle is composed of granulosa cells and oocytes, and the diameter of mouse primary oocytes is reported to be in the range of 50-70 μm (Danny et al. 2021; Nottola et al. 2011; Kohama et al. 2022). The results of this study also showed that within the r-ovarioid, oocytes showed a follicle-like structure surrounded by granulosa cells, and the diameter of the oocytes was in a range similar to previous reports. Furthermore, laminin is reported to be present at the follicular border (Irving-Rodgers and Rodgers 2005; Berkholtz et al. 2006). Similarly, in the present study, laminin was identified around granulosa cells within the r-ovarioid follicle. Additionally, the formation of follicle-like structures in r-ovarioid is closely related to the interaction between the oocyte and granulosa cells, and gap junctions have been reported to mediate this interaction (Dumesic et al. 2015; Alam and Miyano 2020). Furthermore, the anchoring of the N-cadherin-catenin complex to the actin cytoskeleton is associated with the organization and stability of cell-cell contact structures (Makrigiannakis et al. 1999; Mrozik et al. 2018). The immunofluorescence staining observed in this study suggests that the structures within the r-ovarioid may partially reflect the follicle-associated microenvironment beyond simple cell aggregates. Additionally, follicle isolation is performed manually, and the number of follicles recovered during this process is reported to be important for subsequent culture procedures (Hayashi et al. 2017). In this study, follicles could be isolated from all groups, and the proportion of isolated follicles in the 3000:30000 group was relatively higher than that of the control group. This demonstrates that the group size and somatic cell number of r-ovarioids can influence the results observed during follicle isolation. Studies have reported that Figla, Nobox, and Lhx8 are oocyte-specific transcription factors associated with early follicle formation (Wang et al. 2020; Chen et al. 2014; Wu et al. 2023). Additionally, Gdf9 and Bmp15 are involved in bidirectional communication between oocytes and granulosa cells (Alam and Miyano, 2020). In this study, the 3000:30000 group observed changes in expression only in some oocyte-related genes, whereas the 2000:20000 group observed decreased expression in many oocyte-related genes, except for Figla. Foxl2, Wnt4, and Nr2f2, other signals important for ovarian development, have been reported to be involved in somatic cell, stromal cell differentiation, and supporting cell lineage commitment (Ottolenghi et al. 2007; Danti et al. 2025; Rastetter et al. 2014). In addition, Cx43 has been reported in previous studies to be a factor associated with gap junctions between somatic cells and granulosa cells (Sánchez and Smitz 2012). In this study, the expression of these somatic cell-related genes showed a relatively increased pattern in the 2000:20000 group. These differences suggest that the molecular environment associated with oocyte growth and follicle-related structure formation may vary depending on the somatic cell composition conditions within the r-ovarioid. Regulated apoptosis in response to stress has been reported to promote the formation of follicular structures by removing unnecessary cells (Sun et al. 2018; Gao et al. 2020), and autophagy in the ovary prevents excessive cell death (Bhardwaj et al. 2022; Kourtis and Tavernarakis 2009). Conversely, excessive reactive oxygen species can impair mitochondrial function and promote apoptosis, thereby reducing follicular structure formation and oocyte quality (Wang et al. 2021; Prasad et al. 2016). Furthermore, decreased mitochondrial stability due to low Tfam expression may further increase the risk of future mitochondrial dysfunction and apoptosis (Amoushahi et al. 2017). Perp and bcl2 are factors associated with stress response and apoptosis regulation, respectively, and are used as indicators reflecting changes in ovarian status (Filali et al. 2009; Attardi et al. 2000). In the 2000:20000 group in this study, changes in the expression of Bcl2 and Perp, as well as differences in oxidative stress and mitochondrial function indices, were observed. This confirms that there may be differences in the stress-related response patterns exhibited by r-ovarioids depending on somatic cell density. Furthermore, a previous study reported that the Akt1-Foxo3 signaling pathway can ensure the oocyte microenvironment and influence the regulation of oocyte survival and activity (Li et al. 2020). In this study, Akt1 was shown to be progressively reduced as the number of somatic cells decreased. These overall changes suggest that differences in stress response patterns depending on somatic cell density may be associated with differences in follicle formation efficiency and maintenance ability. In conclusion, this study, using an in vitro-generated r-ovarioid system, observed the formation of follicle-like structures and microenvironments in r-ovarioids with reduced somatic cell populations. Furthermore, it suggests that somatic cell density may be associated with oocyte-somatic cell interactions and stress response patterns. However, the analyses presented in this study primarily relied on marker-based expression analysis, and further validation is required to determine whether the observed transcriptional changes directly translate into functional outcomes. Nonetheless, this study provides basic evidence that somatic cell density regulation may influence the structural environment and stress response of r-ovarioid. Statements and Declarations The authors declare no conflict of interest. Acknowledgements We thank all members of Il-Keun Kong's laboratory for their contributions to this effort. Author’s Contributions S-E.L. designed the study, conducted and analyzed the study, and wrote the paper. M.I. designed, reviewed, and guided the study. S.U. assisted with data analysis and experiments. Z.J.G., M.T.K., M-D.J., Y-X.J., M.J.Z., X-F.Y., and S.P. provided the assistance with experiments. I-K.K. wrote the paper and reviewed and supervised the paper. Funding This study was supported partial by the Korean government's National Research Foundation of Korea (NRF) grant (MSIT; No. RS-2023-00208894) and a scholarship from BK21 Four. References Alam MH, Miyano T (2020) Interaction between growing oocytes and granulosa cells in vitro. 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15:21:29","extension":"html","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140753,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/1e903e1341519eca0faa02a2.html"},{"id":100809483,"identity":"16640c99-f06b-4588-b559-3e67dc172b9c","added_by":"auto","created_at":"2026-01-21 15:21:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":239466,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of follicle-like structures observed in each group. The ratio of primordial germ cells (PGCs) to germ stem cells (GSCs) was fixed at 1:10. a) Morphologies of r-ovarioids from each group with reduced somatic cells. Scale bar, 50 μm. b) Number of follicles observed in each group. c) Proportion of observed follicles calculated based on somatic cell count in r-ovarioids (n = 9, *p \u0026lt; 0.05; **p \u0026lt; 0.01; ****p \u0026lt; 0.0001). r-ovarioids, reconstituted ovarioids\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/780b4d6afff8dbccae52887f.png"},{"id":100857898,"identity":"b011d67b-af46-4711-80fc-1711e943d5d7","added_by":"auto","created_at":"2026-01-22 07:23:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":388037,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of oocytes in r-ovarioids. a) Confocal immunofluorescence images of r-ovarioids from small and large groups NR5A1 FITC (green), DDX4 Alexa fluor (light blue). b) DAPI staining image of oocytes isolated from r-ovarioids. Nuclear staining revealed the germinal vesicle (GV) stage, characteristic of a secondary follicle. Scale bar, 50 μm. c) GV oocyte diameter collected from r-ovarioids. d) mRNA expression levels for analyzing genes related to oocyte structural stages and growth (n = 3, *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001). BF, bright field; DAPI, 4′,6-diamidino-2-phenylindole; Figla, filamin A; Nobox, newborn ovary homeobox; Lhx8, Lim homeobox 8; Gdf9, Growth differentiation factor 9; Bmp15, Bone morphogenetic protein 15\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/cdfda0421fcff93ccca0e4ff.png"},{"id":100809485,"identity":"ac5a6e64-52c9-4813-ac8d-1a61b0284b90","added_by":"auto","created_at":"2026-01-21 15:21:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":330858,"visible":true,"origin":"","legend":"\u003cp\u003eObservation and mRNA analysis of somatic cell structure and contact-related markers in r-ovarioids. a) Confocal images of NR5A1 FITC (green) and LAMININ Alexa fluor (light blue) staining. Identification of follicular structures within r-ovarioids. b) RNA expression levels for somatic cell-related gene analysis (n = 3, *p \u0026lt; 0.05; ***p \u0026lt; 0.001). c, d) Confocal images of N-CADHERIN FITC (green), β-CATENIN, and CD44 Alexa fluor (light blue). Scale bar, 50 μm. f) RNA expression levels for stromal cell-related gene analysis (n = 3, *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001). DAPI: 4′,6-diamidino-2-phenylindole; Foxl2, forkhead box L2; Wnt4, wingless-type MMTV integration site family member 4; Afadin, actin filament binding protein; Cx43, connexin 43; Cx37, connexin 37; Nr2f2, nuclear receptor subfamily 2 group f member\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/88d28cdbd597c5fea3fe3792.png"},{"id":100809486,"identity":"20fc3527-a911-4609-9f02-dd8d0150e49a","added_by":"auto","created_at":"2026-01-21 15:21:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":163731,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of apoptosis- and autophagy-associated markers in r-ovarioids. a, b) Confocal images of CASPASE-3 Alexa fluor (light blue) and BECN1 FITC (green). Scale bar, 50 μm. c) RNA expression levels for cell death-associated gene analysis (n = 3, *p \u0026lt; 0.05; ****p \u0026lt; 0.0001). DAPI, 4′,6-diamidino-2-phenylindole; Bax, Bcl2-associated X protein; Bcl2, B-cell leukemia; Perp, P53 apoptosis effector-related to pmp-22\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/3f42111f31bbbe6c0ae3cf52.png"},{"id":100857901,"identity":"a0224436-c00a-40f6-83e0-94680fa57f6d","added_by":"auto","created_at":"2026-01-22 07:23:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":172877,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional stress, surveillance, and follicle maintenance–related markers in r-ovarioids.\u003cstrong\u003e \u003c/strong\u003ea) ROS staining of oocytes collected from r-ovarioids (n = 3, *p \u0026lt; 0.05). b) Mitochondrial membrane potential was detected by JC-1 staining of oocytes collected from r-ovarioids. Green (FITC) signals indicate mitochondria with low membrane potential, and RED (TRITC) signals indicate mitochondria with high membrane potential (n = 3). Scale bar, 50 μm. c) RNA expression levels for mitochondrial-related gene analysis (n = 3, ****p \u0026lt; 0.0001). d) RNA expression levels for cellular surveillance-related gene analysis (n = 3, *p \u0026lt; 0.05; **p \u0026lt; 0.01). e) RNA expression levels for follicle maintenance-related gene analysis (n = 3, ***p \u0026lt; 0.001; ****p \u0026lt; 0.0001). BF, bright field; Tfam, mitochondrial transcription factor A; Brca1, breast cancer 1; Chek2, checkpoint kinase 2; Tap63, tumor protein p63; Foxo3, forkhead box O3; Akt1, Akt serine/threonine kinase 1\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/1af7f4e038228096d0f67971.png"},{"id":102858321,"identity":"43eba47f-bea7-439e-b4b2-2faaeeb7133b","added_by":"auto","created_at":"2026-02-17 15:39:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2169598,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/4c7cbc19-7439-4560-b053-6ba1e659df4d.pdf"},{"id":101202458,"identity":"2a9cc527-5fef-48a0-99eb-faf93d208a2b","added_by":"auto","created_at":"2026-01-27 09:33:48","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9808864,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/a96d17a2d220d4a5ffb38a6a.tif"},{"id":100857830,"identity":"248db87c-f4f8-470f-9c24-6ec2517dc6bd","added_by":"auto","created_at":"2026-01-22 07:23:04","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2031288,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/e2105c80976320bc40544f38.tif"},{"id":100809490,"identity":"582b193c-9e0a-42a7-b07e-2b1f1aa6ecfb","added_by":"auto","created_at":"2026-01-21 15:21:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":168405,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8588693/v1/bcf9bb6a2337af224bda7cc0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of follicle formation and molecular characteristics in reconstituted ovarioids under limited somatic cell conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOogenesis is the process by which primordial germ cells (PGCs) in the ovary develop from primary oocytes into mature oocytes through meiosis, mediated by interactions with gonadal somatic cells (GSCs) within the follicle (Wang et al. 2017; Nagaoka et al. 2021; Wei et al. 2024). During this process, GSCs support the growth of primordial germ cells (PGCs) through their interactions with them, and subsequently differentiate into theca cells and granulosa cells, contributing to the structural framework of the ovary (Zhang et al. 2014). Specifically, follicular somatic cells regulate meiotic arrest and progression, maintaining oocytes in a dormant state at the primordial follicle stage and inducing the resumption of meiosis in specific follicles in response to FSH (Umeno et al. 2022). Furthermore, follicular somatic cells induce follicular attrition and apoptosis during the follicular development cycle, ensuring that only the most viable follicles progress to ovulation (Wang et al. 2023). Therefore, the quality of the microenvironment formed by follicular somatic cells plays a key role in regulating ovarian structure, oocyte maturation, and overall reproductive success. Because follicles constitute a limited ovarian reserve at birth, early follicular formation and the stability of the microenvironment are crucial for maintaining female reproductive function (Zhang and Liu 2015).\u003c/p\u003e\n\u003cp\u003eBased on germ cell-somatic cell interactions, in vitro oogenesis research in recent years has focused on preserving female fertility and enhancing our understanding of oogenesis mechanisms (Clarke 2018; Wang et al. 2017). Early research utilized oocyte-granulosa cell complexes isolated from newborn ovaries, which were cultured in two-dimensional (2D) in tissue culture well inserts to induce in vitro maturation (O\u0026apos;Brien et al. 2003; Jin et al. 2010). Subsequent development of three-dimensional (3D) culture systems has supported prefollicular formation while better mimicking the in vivo follicle structure (Khunmanee and Park 2022). These systems mimic the extracellular matrix and promote gap junction formation between primordial follicle cells (PGCs) and germline stem cells (GSCs), resulting in superior follicle formation compared to two-dimensional (2D) systems (Green and Shikanov 2016). Advances in in vitro oogenesis techniques have also included the cultivation of neonatal mouse ovaries. For example, fertile oocytes were produced by culturing female gonads isolated from embryonic day 12.5 (E12.5) mouse embryos (Eppig and O\u0026rsquo;Brien 1996; Morohaku et al. 2017). Subsequently, ovaries were formed by reaggregating primordial germ cell-like cells (PGCLCs) differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) with germ cell stem cells (GSCs) isolated from mouse E12.5. This is called a reconstituted ovary (r-ovary), and it is proposed as an experimental platform that enables follicle formation and oocyte development in vitro (Hayashi et al. 2012; Hikabe et al. 2016; Hayashi et al. 2017).\u003c/p\u003e\n\u003cp\u003eHowever, oogenesis from mouse reconstituted ovaries remains limited by the difficulty in obtaining sufficient numbers of E12.5 GSCs, which provide the necessary environment for PGC growth (Wang et al. 2021). Typically, 5,000 PGCLCs and 50,000 GSCs are required to generate a reconstituted ovary. This requires the removal of residual PGCs and endothelial cells from approximately 2.5 female gonads, a labor-intensive and inefficient process (Hikabe et al. 2016; Hayashi et al. 2017; Sosa et al. 2023). This challenge is particularly pronounced in species such as humans, where fetal GSCs cannot be obtained (Hayashi et al. 2017). The differentiation process to replace this is expected to require approximately 75,000\u0026ndash;100,000 GSCs (Yoshino et al. 2021). However, stable and scalable culture systems for GSC-derived cells remain limited. These limitations significantly impede the large-scale production of artificial ovaries. Nevertheless, complete elimination of GSCs from reconstituted ovaries is currently impossible because interactions between oocytes and somatic cells within the ovary are crucial for oocyte development (Lo et al. 2019; Vo and Kawamura 2021). Therefore, to address this issue, this study constructed structurally stable reconstituted ovarioids (r-ovarioids) with minimal GSCs and evaluated the feasibility of follicle formation under limited somatic cell conditions.\u003c/p\u003e\n\u003cp\u003eThe results demonstrated that follicle-like structures were observed in r-ovarioids with reduced somatic cell counts, partially reproducing the microenvironment derived from somatic cells. Conversely, significant differences in the expression of follicle- and stress-related molecules were observed depending on the number of somatic cells. These results demonstrate that follicle formation is possible even under limited somatic cell conditions and are expected to provide basic data for optimizing conditions for reconstructed ovarian models.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch3\u003eAll reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.\u003c/h3\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Gyeongsang National University (Approval Number: GNU-240605-M0119). Eight-week-old CD-1 (ICR) mice obtained from Coretech (Seoul, Republic of Korea) were used for the experiments. The animals were maintained under a 12-h light/dark cycle with ad libitum access to food and water. Female mice were used on E12.5, based on the observation of vaginal plugs following natural mating with CD-1 (IGR) males. The day of vaginal plug detection was designated as E0.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGonad isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn E12.5 pregnant mouse was euthanized by cervical dislocation, and embryos were carefully collected from the uterus. Embryos were washed several times with saline solutions to remove residual maternal tissues. Gonadal ridges were isolated from each embryo using a stereomicroscope. The gonads were separated from the surrounding mesonephros using a fine needle. Isolated gonads were dissociated into single-cell suspensions using 0.05% (w/v) trypsin-EDTA (Gibco BRL, Cat. No. 25300-062, USA) at 37\u0026deg;C. The dissociated cells were washed with a Leibovitz 15 medium (L-15; Cat. No. L5520) to neutralize trypsin and subsequently used for further experimental procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMACS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMACS was performed according to the manufacturer\u0026apos;s instructions (Miltenyi Biotec, Germany). Germline cells dissociated using 0.05% (w/v) trypsin-EDTA were resuspended in MACS buffer, which consisted of MACS\u0026reg; BSA Stock Solution (10% bovine serum albumin in phosphate-buffered saline (PBS), Cat. No. 130-091-376) and 0.5mM EDTA (Cat. No. E7889) in Dulbecco\u0026rsquo;s PBS (DPBS; Gibco BRL, Cat. No. 14190-250). The cell suspension was incubated on ice for 15 min with magnetic bead-conjugated antibodies, Anti-SSEA1 (Miltenyi, Cat. No. 130-094-530) and Anti-CD31(Miltenyi, Cat. No. 130-097-418). The mixture was then processed through MS columns (Magnetic Separation column) using a mini-MACS separation unit, following the manufacturer\u0026rsquo;s instructions. Non-magnetically retained cells were classified as PGCs, whereas magnetically retained cells were classified as GSCs. Cell counts were determined using a hemocytometer, and cell concentrations were adjusted as required for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReaggregation of PGCs and GSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter adjusting the cell ratio to 1:10, the cells were reaggregated into three-dimensional cell aggregates under suspension culture conditions using 96-well U-shaped low-binding culture plates (Thermo Fisher Scientific, catalog no. 174925, USA). The cells were then cultured in a controlled humidity atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and 37.5\u0026deg;C for 3 days. To promote PGC expression, 0.3 \u0026micro;M retinoic acid (Cat. No. R2625) was added to the GK15 medium. The GK medium was prepared by mixing the following components: Glasgow\u0026apos;s Modified Eagle Medium (GMEM; Gibco BRL, Cat. No. 11710035) with 15% (v/v) KnockOut\u0026trade; Serum Replacement (Gibco BRL, Cat. No. 10828010), 1\u0026times; MEM Non-Essential Amino Acids (Gibco BRL, Cat. No. 11140-050), 1 mM sodium pyruvate (Gibco BRL, Cat. No. 11360-070), 2 mM GlutaMAX\u0026trade; supplements (Gibco BRL, Cat. No. 35050061), 1\u0026times; penicillin-streptomycin (Cat. No. P4333), and 25.7 mM \u0026beta;-mercaptoethanol (Gibco BRL, Cat. No. 21985023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro differentiation (IVDi)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePGC and GSC differentiation was promoted using the IVDi culture method (Hayashi et al. 2017). Cultures were conducted in Transwell-COL membrane insert wells (Corning, Cat. No. 3493, NY, USA), to maintain the pre-established three-dimensional structure, with media changes every 2 days. Two types of culture media were used sequentially. MEM-based IVDi medium was used for the initial 4 days. This medium was prepared by mixing 2% (v/v) fetal bovine serum (FBS; Gibco BRL, Cat. No.16000-044), 0.15 nM ascorbic acid stock solution (TCI America, Cat. No. G0394), 2 mM GlutaMax, 1\u0026times; penicillin-streptomycin, and 14.3 mM \u0026beta;-mercaptoethanol in MEM Alpha (Thermo Fisher Scientific, Cat. No. 12571-063). After four days, the cultures were maintained in StemPro-34-based IVDi medium. This medium was prepared by mixing, 10% (v/v) FBS, 0.15 nM ascorbic acid stock solution, 2 mM GlutaMax, 1\u0026times;penicillin-streptomycin, and 14.3 mM \u0026beta;-mercaptoethanol in StemPro-34 SFM (Gibco BRL, Cat. No. 10639-011). The culture continued for 21 days. To prevent the formation of polycystic follicles, estrogen inhibitors were added from days 7 to 11 of the culture. The estrogen inhibitors were prepared by dissolving 12.14 mg of ICI 182,780 (Estrogen receptor antagonist, Tocris, Cat. No. 1047) in DMSO (Cat. No. D8418) to create a 20 mM stock solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of secondary follicles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 21 days of culture,\u0026nbsp;the r-Ovaries\u0026nbsp;were dissected to isolate secondary follicles using a tungsten needle. Follicles\u0026nbsp;in which the oocyte had ruptured, or the follicular structure had been damaged were excluded. After isolation, secondary follicles were evaluated in two ways: based on starting cell count (compared to PGC count) and based on constituent cells (compared to somatic cell count).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJC-1 staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mitochondrial membrane potential (\u0026Psi;) of oocytes was analyzed using JC-1 staining. After washing the live oocytes with PVA-PBS, 2.0 \u0026mu;g/mL of JC-1 was diluted in DPBS and incubated with the oocytes at 37.5\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 30 min. After incubation, the process should be performed in a darkroom, and the cells were washed with 2% (v/v) PBS-PVA and stained with DAPI for 5 min. Afterwards, it was washed again with 2% (v/v) PBS-PVA and observed using a laser scanning confocal microscope. The signal intensities of mitochondrial monomers (green) and aggregates (red) were measured using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH2DCFDA was used to measure ROS levels. Oocytes isolated from secondary follicles and having their cumulus cells removed were placed in a PBS solution containing 1 nM H2DCFDA and incubated for 30 min at 37.5\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Afterwards, they were washed with PBS and photographed using an Olympus IX71 epifluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of complementary DNA (cDNA) and real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe r-Ovaries from IVDi culture on days 0 and 21 were washed three times with nuclease-free water, placed in a 1.5-mL tube containing 30 \u0026mu;L of nuclease-free water, and fresh-frozen in liquid nitrogen. Samples were stored at \u0026minus;80\u0026deg;C until processing. Total mRNA was extracted using a\u0026nbsp;PicoPure RNA isolation kit (Thermo Fisher Scientific, Cat. No. KIT0204) according to the manufacturer\u0026apos;s instructions. The concentration and purity of the mRNA were verified using a NanoDrop 2000/2000c spectrophotometer. The isolated mRNA was reverse transcribed into single-stranded cDNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Cat. No.1708891, Hercules, CA, USA). Gene-specific primers were designed using the Primer3Plus software (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) and are listed in Online Resource 1. The primers synthesized based on mRNA sequences were purchased from Macrogen (Seoul, Republic of Korea). Quantitative real-time PCR (qRT-PCR) was conducted on a CFX98 instrument (Bio-Rad Laboratories) using a 10-\u0026micro;L reaction mixture comprising 1\u0026times; iQ SYBR Green Supermix (Bio-Rad Laboratories, Cat. No. 170-8882AP), 3 \u0026mu;L of diluted DNA, and 0.2 mM of each primer. All cDNA samples were normalized to the housekeeping gene, \u003cem\u003eGAPDH\u003c/em\u003e, whose expression remained constant across all samples. Transcript quantification was performed using the \u0026Delta;\u0026Delta;Ct method, with normalized to the average \u003cem\u003eGAPDH\u003c/em\u003e expression. The qRT-PCR thermal profile included an initial denaturation at 95\u0026deg;C for 3 min, followed by 44 cycles of 95\u0026deg;C for 15 s, 56\u0026deg;C for 20 s, and 72\u0026deg;C for 30 s, with a final extension at 72\u0026deg;C for 5 min. The coefficients of intra- and interassay variance for each gene were calculated as follows: (standard deviation/mean) \u0026times; 100.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe r-Ovaries from IVDi cultures on day 21 were fixed with 4% paraformaldehyde at 4\u0026deg;C for 30 min and prepared for immunofluorescence staining. The r-Ovaries were washed three times with 0.2% (w/v) polyvinyl alcohol in PBS (PVA-PBS) for 10 min each. To enhance permeability, cells were treated with proteinase K for 5 min, followed by three washes. The samples were then incubated in a blocking solution (5% BSA in PVA-PBS) at 25\u0026deg;C for 90 min. After blocking, the r-Ovaries were incubated overnight at 4\u0026deg;C with primary antibodies (Online Resource 2). The samples were then washed three times with PVA-PBS and incubated with fluorescently labeled secondary antibodies (fluorescein isothiocyanate conjugated (FITC), tetramethylrhodamine isothiocyanate, and Alexa Fluor\u0026reg; 647; Santa Cruz Biotechnology, Dallas, Texas, USA) at 25\u0026deg;C for 90 min. For nuclear staining, the r-Ovaries were treated with DAPI (Thermo Fisher Scientific, Cat. No. 62248) at 25\u0026deg;C for 5 min. After the final wash, all r-Ovaries were mounted on glass slides using Anti-Fade Fluorescence Mounting Medium - Aqueous, Fluoroshield (Abcam, Cat. No. ab104135). Confocal imaging was performed using a laser scanning confocal microscope (Fluoview FV 1000, Olympus, Tokyo, Japan) and fluorescence intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses of follicular development and gene expression levels between large and small groups were conducted using one-way ANOVA and t-tests in GraphPad Prism 6 (Graph Pad Software, San Diego, CA, USA), followed by Tukey\u0026rsquo;s post-hoc test for multiple pairwise comparisons between groups. Each experiment was performed in triplicate. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, with significance defined at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Immunofluorescence and integral optical densities were quantified using ImageJ software and GraphPad Prism 6.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorphology and follicle of r-ovarioids with reduced somatic cell counts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, structures identified based on morphological characteristics in r-ovarioids were referred to as follicle-like structures, whereas follicles isolated and quantified were defined as follicles. In aggregates that were too small, the 3D structure collapsed when transferred to insert wells (Online Resource 3). From the 1000:10000 group, the 3D structure was maintained, but normal follicle-like structures were not observed after culture (Fig. 1a). On the other hand, in the 2000:20000 group, follicular morphology began to appear from day 14, and follicle-like structure was clearly observed on day 21 of culture. The 3000:30000 group also showed similar patterns, and the structure was maintained throughout the culture period, similar to the control group, 5000:50000. As expected, the control group observed the largest number of follicle-like structures (Fig. 1b), and the number of observed structures decreased as somatic cells decreased. After culture, structures resembling secondary follicles were observed in r-ovarioids, and morphologically maintained secondary follicles could be isolated from r-ovarioids. After directly isolating secondary follicles from r-ovarioids, the ratio of recovered secondary follicles was calculated based on the number of somatic cells (Fig. 1c). The 3000:30000 group showed a relatively high ratio, and the 2000:20000 group showed a similar level to the control group. In addition, when normalized based on the number of PGCs and analyzed, similar results were shown to the analysis based on the number of somatic cells (0.51 \u0026plusmn; 0.10 vs. 0.71 \u0026plusmn; 0.20 vs. 0.56 \u0026plusmn; 0.25; p \u0026lt; 0.05; Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Comparison of r-ovarioid outcomes across PGC:GSC cell count ratios\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3213%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell ratios of PGCs: GSCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.0148%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of\u0026nbsp;\u003cbr\u003e\u0026nbsp;r-ovarioids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.0033%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of total cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.8847%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of PGCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.0527%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of presumed secondary follicles\u0026nbsp;\u003cbr\u003e (% \u0026plusmn; SE)\u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.7232%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of isolation secondary follicles\u0026nbsp;\u003cbr\u003e (% \u0026plusmn; SE)\u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3213%;\"\u003e\n \u003cp\u003e5000:50000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.0148%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.0033%;\"\u003e\n \u003cp\u003e495000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.8847%;\"\u003e\n \u003cp\u003e45000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.0527%;\"\u003e\n \u003cp\u003e390 (0.87 \u0026plusmn; 0.10)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.7232%;\"\u003e\n \u003cp\u003e228 (0.51 \u0026plusmn; 0.10)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3213%;\"\u003e\n \u003cp\u003e3000:30000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.0148%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.0033%;\"\u003e\n \u003cp\u003e297000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.8847%;\"\u003e\n \u003cp\u003e27000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.0527%;\"\u003e\n \u003cp\u003e327 (1.21 \u0026plusmn; 0.08)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.7232%;\"\u003e\n \u003cp\u003e191 (0.71 \u0026plusmn; 0.20)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.3213%;\"\u003e\n \u003cp\u003e2000:20000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.0148%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.0033%;\"\u003e\n \u003cp\u003e198000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.8847%;\"\u003e\n \u003cp\u003e18000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.0527%;\"\u003e\n \u003cp\u003e194 (1.08 \u0026plusmn; 0.18)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.7232%;\"\u003e\n \u003cp\u003e100 (0.56 \u0026plusmn; 0.25)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eRates were calculated by normalizing the number of presumed or isolated secondary follicles to the number of PGCs.\u003c/p\u003e\n\u003cp\u003eThree replicates were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 with different superscripts indicates the significant difference.\u003c/p\u003e\n\u003cp\u003er-ovarioids, reconstituted ovarioids; PGCs, primordial germ cells; 5000:50000, reconstructed ovarioids produced from a group of 5000:50000; 3000:30000, reconstructed ovarioids produced from a group of 3000:30000; 2000:20000, Reconstructed ovarioids produced from a group of 2000:20000.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePresence of secondary follicles and oocytes in r-ovarioids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvaluation of the intrafollicular oocyte characteristics observed in r-ovarioids with reduced somatic cells revealed that all r-ovarioids expressed DDX4 (a germ cell marker), which was surrounded by NR5A1 (a germ cell somatic marker), suggesting the presence of follicle-like structures (Fig. 2a). DAPI staining revealed that the oocyte nuclei were at the germinal vesicle stage, showing an appearance consistent with the characteristics of secondary follicles (Fig. 2b). Furthermore, the diameters of oocytes isolated from secondary follicles ranged from 50 to 70 \u0026micro;m (62.73 \u0026plusmn; 2.44 vs. 61.15 \u0026plusmn; 1.78 vs. 59.89 \u0026plusmn; 4.92; p \u0026lt; 0.05; Fig. 2c). Additionally, when we examined the mRNA expression of genes (Figla, Nobox, Lhx8, Gdf9, Bmp15) related to oocyte-specific transcriptional regulation and growth in r-ovarioid (Fig. 2d), a significant decrease in expression was observed only in Lhx8 and Bmp15 in the 3000:30000 group (p \u0026lt; 0.05, p \u0026lt; 0.01), whereas other genes did not show statistically significant differences. However, in the 2000:20000 group, except for Figla, the remaining genes showed significantly lower expression levels than the control group (p \u0026lt; 0.05, p \u0026lt; 0.001, p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural organization of somatic cells and expression of junction-related markers in r-ovarioids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the spatial characteristics of somatic cells and follicle-like structures in reduced r-ovarioids, LAMININ immunostaining was performed. Round follicle-like structures surrounded by LAMININ were observed in all r-ovarioids (Fig. 3a). The mRNA expression of early somatic differentiation-related factors (Foxl2, Wnt4) showed a similar trend to the control group in the 3000:30000 group (Fig. 3b). In contrast, the 2000:20000 group showed a significantly increased expression level compared to the control group, although the number of somatic cells was reduced (p \u0026lt; 0.05, p \u0026lt; 0.001). In addition, immunostaining for cell adhesion proteins (N-CADHERIN, CD 44) was performed to visualize the cell-cell contact patterns of somatic cells located around the follicle-like structures in r-ovarioids (Fig. 3c). As a result, N-CADHERIN signals were observed adjacent to the follicle-like structures, and \u0026szlig;-CATENIN, which stabilizes the interaction of N-CADHERIN, was also observed in all groups. Additionally, CD44 was also expressed on the surface of r-ovarioids in all groups. Analysis of the mRNA expression levels of cell junction-related genes (afadin, Cx43, Cx37) showed that only the expression of Cx43 was significantly increased in both the 3000:30000 group and the 2000:20000 group (p \u0026lt; 0.01, p \u0026lt; 0.001, respectively; Fig. 3e). Nr2f2, a GSC-derived stromal cell gene, was significantly increased only in 2000:20000 group compared to the control group (p \u0026lt; 0.05; Fig. 3f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of apoptosis- and autophagy-associated markers in r-ovarioids\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To evaluate cellular stress responses in r-ovarioid, the expression of apoptosis and autophagy-related markers was analyzed. Immunofluorescence signals of CASPASE-3 and BECN1 were observed within r-ovarioids in all experimental groups (Fig. 4a, b). Comparison of mRNA expression levels to determine the level of apoptosis (Bax, Bcl2, Perp) revealed that the 3000:30000 group exhibited similar expression levels to the control group (Fig. 4c). In contrast, the 2000:20000 group observed increased expression levels in Bcl2 and Perp (p \u0026lt; 0.05, p \u0026lt; 0.0001). These results demonstrate differences in the expression patterns of apoptosis-related genes under various conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObservation of functional stress-related changes in r-ovarioids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the functional stress state within r-ovarioid, the level of intracellular ROS production and mitochondrial membrane potential were analyzed. ROS levels significantly increased in the 2000:20000 group compared to the control group (Fig. 5a), and JC-1 staining results showed no differences across all groups (Fig. 5b). However, analysis of Tfam mRNA expression, an indicator of mitochondrial health, revealed decreased expression in the 2000:20000 and 3000:30000 groups compared to the control group (Fig. 5c). Furthermore, analysis of the expression of genes involved in DNA damage surveillance (Brca1, Chek2, Tap63) compared to the control group revealed that Brca1 and Chek2 increased in the 2000:20000 group, Chek2 decreased in the 3000:30000 group, and Tap63 showed no significant differences across all groups (Fig. 5d). In addition, comparative analysis of the Foxo3-Akt1 axis related to the regulation of follicular status showed that the expression of Foxo3 did not differ significantly in all groups, but the expression of Akt1 was significantly reduced in both groups compared to the control group (Fig. 5e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA limited ovarian pool of follicles and finite number of oocytes present substantial challenges in sustaining the reproductive potential (Chen et al.2014). To overcome this limitation, extensive studies have focused on oogenesis (Hayashi et al. 2017; Morohaku et al. 2017). Despite these advances, generating r-Ovaries requires a substantial number of GSCs, with approximately 1.5 times more GSCs required for applications after cryopreservation (Yoshino et al. 2021; Sosa et al. 2023). Even for differentiating large numbers of GSCs, non-mechanized protocols still pose challenges in meeting these requirements. However, because germ cells rapidly undergo apoptosis without support from GSCs, establishing efficient control of GSC numbers is a key area of need (Eppig 2001; Shen et al.2006; Dumesic et al. 2015). Therefore, this study compared r-ovaroids produced under conditions requiring a reduced GSC requirement to evaluate the characteristics of an in vitro culture system that can be utilized for oogenesis research.\u003c/p\u003e\n\u003cp\u003eResearch on ovarian reconstitution and follicular development has progressed in stages. Previous studies have reported that follicle-like structures can be obtained under conditions where 5,000 primordial germ cells (PGCs) and 50,000 germ stem cells (GSCs) are aggregated (Hikabe et al. 2016; Hayashi et al. 2017). Additionally, structures classified as primordial follicles are generally known to be surrounded by fewer than 10 granulosa cells in mice and 4–10 granulosa cells in cats and sheep (Lundy et al. 1999; Ariel et al. 2016; Wei et al. 2024). Therefore, in this study, r-ovarioids were cultured under conditions that allowed observation of follicle-like structures while maintaining the previously established cell ratio of 1:10. Initial attempts to form r-ovarioid with relatively small cell numbers (e.g., 1:10) resulted in difficulty in distinguishing cell aggregation, and limited observation of follicle-like structures in smaller groups (e.g., 10:100, 20:200, 50:500). This suggests that a certain number of cells may be required for r-ovarioid formation. In addition, according to previous studies, the composition of the follicle is composed of granulosa cells and oocytes, and the diameter of mouse primary oocytes is reported to be in the range of 50-70 μm (Danny et al. 2021; Nottola et al. 2011; Kohama et al. 2022). The results of this study also showed that within the r-ovarioid, oocytes showed a follicle-like structure surrounded by granulosa cells, and the diameter of the oocytes was in a range similar to previous reports. Furthermore, laminin is reported to be present at the follicular border (Irving-Rodgers and Rodgers 2005; Berkholtz et al. 2006). Similarly, in the present study, laminin was identified around granulosa cells within the r-ovarioid follicle. Additionally, the formation of follicle-like structures in r-ovarioid is closely related to the interaction between the oocyte and granulosa cells, and gap junctions have been reported to mediate this interaction (Dumesic et al. 2015; Alam and Miyano 2020). Furthermore, the anchoring of the N-cadherin-catenin complex to the actin cytoskeleton is associated with the organization and stability of cell-cell contact structures (Makrigiannakis et al. 1999; Mrozik et al. 2018). The immunofluorescence staining observed in this study suggests that the structures within the r-ovarioid may partially reflect the follicle-associated microenvironment beyond simple cell aggregates. Additionally, follicle isolation is performed manually, and the number of follicles recovered during this process is reported to be important for subsequent culture procedures (Hayashi et al. 2017). In this study, follicles could be isolated from all groups, and the proportion of isolated follicles in the 3000:30000 group was relatively higher than that of the control group. This demonstrates that the group size and somatic cell number of r-ovarioids can influence the results observed during follicle isolation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudies have reported that Figla, Nobox, and Lhx8 are oocyte-specific transcription factors associated with early follicle formation (Wang et al. 2020; Chen et al. 2014; Wu et al. 2023). Additionally, Gdf9 and Bmp15 are involved in bidirectional communication between oocytes and granulosa cells (Alam and Miyano, 2020). In this study, the 3000:30000 group observed changes in expression only in some oocyte-related genes, whereas the 2000:20000 group observed decreased expression in many oocyte-related genes, except for Figla. Foxl2, Wnt4, and Nr2f2, other signals important for ovarian development, have been reported to be involved in somatic cell, stromal cell differentiation, and supporting cell lineage commitment (Ottolenghi et al. 2007; Danti et al. 2025; Rastetter et al. 2014). In addition, Cx43 has been reported in previous studies to be a factor associated with gap junctions between somatic cells and granulosa cells (Sánchez and Smitz 2012). In this study, the expression of these somatic cell-related genes showed a relatively increased pattern in the 2000:20000 group. These differences suggest that the molecular environment associated with oocyte growth and follicle-related structure formation may vary depending on the somatic cell composition conditions within the r-ovarioid.\u003c/p\u003e\n\u003cp\u003eRegulated apoptosis in response to stress has been reported to promote the formation of follicular structures by removing unnecessary cells (Sun et al. 2018; Gao et al. 2020), and autophagy in the ovary prevents excessive cell death (Bhardwaj et al. 2022; Kourtis and Tavernarakis 2009). Conversely, excessive reactive oxygen species can impair mitochondrial function and promote apoptosis, thereby reducing follicular structure formation and oocyte quality (Wang et al. 2021; Prasad et al. 2016). Furthermore, decreased mitochondrial stability due to low Tfam expression may further increase the risk of future mitochondrial dysfunction and apoptosis (Amoushahi et al. 2017). Perp and bcl2 are factors associated with stress response and apoptosis regulation, respectively, and are used as indicators reflecting changes in ovarian status (Filali et al. 2009; Attardi et al. 2000). In the 2000:20000 group in this study, changes in the expression of Bcl2 and Perp, as well as differences in oxidative stress and mitochondrial function indices, were observed. This confirms that there may be differences in the stress-related response patterns exhibited by r-ovarioids depending on somatic cell density. Furthermore, a previous study reported that the Akt1-Foxo3 signaling pathway can ensure the oocyte microenvironment and influence the regulation of oocyte survival and activity (Li et al. 2020). In this study, Akt1 was shown to be progressively reduced as the number of somatic cells decreased. These overall changes suggest that differences in stress response patterns depending on somatic cell density may be associated with differences in follicle formation efficiency and maintenance ability.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study, using an in vitro-generated r-ovarioid system, observed the formation of follicle-like structures and microenvironments in r-ovarioids with reduced somatic cell populations. Furthermore, it suggests that somatic cell density may be associated with oocyte-somatic cell interactions and stress response patterns. However, the analyses presented in this study primarily relied on marker-based expression analysis, and further validation is required to determine whether the observed transcriptional changes directly translate into functional outcomes. Nonetheless, this study provides basic evidence that somatic cell density regulation may influence the structural environment and stress response of r-ovarioid.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all members of Il-Keun Kong\u0026apos;s laboratory for their contributions to this effort.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS-E.L. designed the study, conducted and analyzed the study, and wrote the paper. M.I. designed, reviewed, and guided the study. S.U. assisted with data analysis and experiments. Z.J.G., M.T.K., M-D.J., Y-X.J., M.J.Z., X-F.Y., and S.P. provided the assistance with experiments. I-K.K. wrote the paper and reviewed and supervised the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported partial by the Korean government\u0026apos;s National Research Foundation of Korea (NRF) grant (MSIT; No. RS-2023-00208894) and a scholarship from BK21 Four.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlam MH, Miyano T (2020) Interaction between growing oocytes and granulosa cells in vitro. 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However, efficient production of reconstructed ovaries remains technically limited by the requirement for securing a large number of gonadal somatic cells. Therefore, this study evaluated the feasibility of follicle formation under limited somatic cell conditions by forming ovarioids with a reduced number of somatic cells. The formation of follicle-like structures was confirmed using immunofluorescence analysis, and the expression of molecular markers related to apoptosis, autophagy, mitochondrial stability, and stress responses was analyzed. As a result, it was observed that the groups with a reduced number of gonad somatic cells (2000:20000 and 3000:30000) formed follicle-like structures similar to the control group. Furthermore, oocytes observed under all conditions ranged in diameter from 50 to 70 \u0026micro;m, corresponding to the primary oocyte stage. Additionally, it was observed that the microenvironment was partially reproduced through staining of LAMININ, N-CADHERIN, and CD44. However, as the number of somatic cells decreased, differences were observed in the expression patterns of follicle-related and stress-related molecules. In particular, compared to the control group, the 2000:20000 group showed a significant decrease in follicle-related factors (Nobox, Lhx8, Gdf9, Bmp15). These results suggest that the number of somatic cells and the composition of the microenvironment in a reconstructed ovarian model can affect the qualitative environment for follicle formation, providing a basic basis for optimizing the conditions for ovarian reconstitution.\u003c/p\u003e","manuscriptTitle":"Comparison of follicle formation and molecular characteristics in reconstituted ovarioids under limited somatic cell conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 15:21:23","doi":"10.21203/rs.3.rs-8588693/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f319b31-6daa-46d2-961c-b5d1d86280ac","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T15:39:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 15:21:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8588693","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8588693","identity":"rs-8588693","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}