O-157 Micro-Computed Tomography of the adult mouse ovary: an in-silico 3D reconstruction of folliculogenesis

Study question Which are the spatial dynamics of follicles recruitment and growth inside the ovary? Summary answer 3D micro-Computed Tomography (microCT) shows a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. What is known already In the mouse ovary, folliculogenesis progresses from the primordial type 1 (T1) to the fully-grown T8 follicle. Most of our knowledge of the folliculogenetic process has been obtained by disaggregating the ovary into its functional units (i.e., follicles and oocytes), thus losing the complexity of the whole histo-functional context. To date, few studies employed 3D imaging approaches to gain information on the inside 3D ovary organisation. MicroCT is the only technique that combines a high spatial resolution (down to ∼1 µm) with the production of a true 3D organ reconstruction, with cubic voxels and isotropic resolution. Study design, size, duration Three ovaries of three different adult mice were treated with the contrast agent and then imaged with microCT. A typical experiment required a total of 35 man/h from ovaries isolation to completion of X-ray scanning, and 24 man/h for follicles classification and mapping. Participants/materials, setting, methods Three ovaries of three different 8-week-old CD1 mice were fixed in 4% Paraformaldehyde and treated with Lugol’s solution for 3 hr at RT. Ovaries were scanned with Skyscan 1172 (Bruker) using a 1.5 µm/pixel resolution. MicroCT sections were processed with Fiji ImageJ (NIH), and 3D rendering of follicles and blood vessels were obtained with Avizo-9 (Thermo Fisher Scientific). ANOVA and Bonferroni post-­hocstatistical analyses were performed with RStudio, considering data significantly different when p < 0.05. Main results and the role of chance Using microCT we built the first in silico3D reconstruction of the tiny mouse ovary, identifying, mapping and counting follicles,from pre-antral secondary T4 (53.2 + 12.7 µm in diameter) to fully-grown antral T8 (321.0 + 21.3 µm), and the corpora lutea.MicroCTbrought up the main functional compartments of the growing follicle, i.e., granulosa and cumulus cells, the antrum, the zona pellucida, and the oocyte with its nucleus. Instead, primordial and primary follicles (T1–T3) could not be observed, perhaps due to the reduced size of their enclosed oocyte and to the absence of a well-formed zona pellucida around the germ cell. In addition, our analysis allowed the visualisation and 3D modelling of the main ovarian vasculature, from the largest vessel that enters the organ at the hilum site (∼150 µm size in diameter)to smaller branches present in the medulla region (∼35 µm). These results show that each of the eight ovarian sectors, virtually segmented along the dorsal-ventral axis,houses an equal number of each follicle type, suggesting a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. Limitations, reasons for caution To strengthen the results, the number of ovaries/individuals analysed should be increased. Wider implications of the findings This 3D mapping of follicles and vessels could contribute our understanding of folliculogenesis dynamics, not only under normal conditions, but also during ageing, after hormones or drugs administration, or in the presence of ovarian pathologies. Trial registration number not applicable

Novel circGFRα1 Promotes Self-Renewal of Female Germline Stem Cells Mediated by m6A Writer METTL14

Circular RNAs (circRNAs) play important roles in the self-renewal of stem cells. However, their significance and regulatory mechanisms in female germline stem cells (FGSCs) are largely unknown. Here, we identified an N6-methyladenosine (m6A)-modified circRNA, circGFRα1, which is highly abundant in mouse ovary and stage-specifically expressed in mouse FGSC development. Knockdown of circGFRα1 in FGSCs significantly reduced their self-renewal. In contrast, overexpression of circGFRα1 enhanced FGSC self-renewal. Mechanistically, circGFRα1 promotes FGSC self-renewal by acting as a competing endogenous RNA (ceRNA) that sponges miR-449, leading to enhanced GFRα1 expression and activation of the glial cell derived neurotrophic factor (GDNF) signaling pathway. Furthermore, circGFRα1 acts as a ceRNA based on METTL14-mediated cytoplasmic export through the GGACU motif. Our study should help to understand the mechanisms regulating germ cell development, add new evidence on the mechanism of action of circRNA, and deepen our understanding of the development of FGSCs.

The Effects of Ovarian Encapsulation on Morphology and Expression of Apoptosis-Related Genes in Vitrified Mouse Ovary

Background: The purpose of this study was to determine the effects of alginate hydrogel as a capsule to protect the ovary against possible detrimental effects of vitrification and warming on morphology and expression of apoptosis-related genes in the mouse ovary. Methods: In this experimental study, the ovaries from twenty-five female 8-week-old mice were divided into five groups of non-vitrified ovaries, vitrified ovaries, ovaries that were encapsulated with concentrations of 0.5, 0.75 and 1% of alginate hydrogel. The morphological study was performed using hematoxylin and eosin staining. Expression levels of apoptosis-associated genes were quantified in each group by real-time RT-PCR. The one-way ANOVA and post hoc test were used to analyze the data and values of p<0.05 were considered statistically significant. Results: The results of follicle count showed that the mean of total follicles in all groups was not significantly different. The average number of atretic follicles in vitrified and experimental groups significantly increased in comparison with the non-vitrified group (p=0.001). The results of the evaluation of apoptosis-related genes showed that the ratio of BAX/BCL-2 in experimental groups 1 and 2 was significantly higher than the vitrified group and experimental group 3 (p=0.000). The expression level of caspase 3 gene was not significantly different among all groups. Conclusion: Ovarian encapsulation with used concentrations of alginate hydrogel failed to improve the morphology and molecular aspects of follicles and it was not able to better preserve the intact follicles of vitrified ovaries. However, morphological and molecular findings appear to improve with increasing alginate hydrogel concentration.