Characterization of isolated late preantral and early antral equine ovarian follicles.

ABSTRACT: Understanding how to effectively isolate a large number of healthy follicles is essential for increasing success rates in ARTs, such as in vitro culture and cryopreservation. Late secondary (preantral) and early tertiary (antral) follicles isolated from the intermediary portion of equine ovarian tissue sliced at two different thicknesses (0.25 vs 0.50 mm) were characterized according to regional distribution (dorsal, medial, and ventral), morphofunctional features, and epigenetic profiles. After isolation via microdissection, follicles were evaluated for membrane integrity (trypan blue), viability (propidium iodide and Hoechst 33342), mitochondrial membrane potential (JC-1), reactive oxygen species (ROS; H2DCFDA), and histone trimethylation patterns (H3K4me3 and H3K9me3). Regardless of the ovarian tissue slice thickness or region, all isolated follicles had intact membranes, confirming the effectiveness of mechanical microdissection. The key findings from this study reveal that i) recovery of preantral and antral follicles within different ovarian regions is influenced by tissue slicing thickness; ii) the abundance of recoverable follicles varies among the ovarian regions; iii) preantral and antral follicle viability and ROS levels remain consistent between developmental stages, irrespective of tissue slicing thickness, although mitochondrial membrane potential is influenced by slicing thickness; iv) in antral follicles, viability and ROS levels vary depending on location within the ovarian regions; and v) epigenetic markers (H3K4me3 and H3K9me3) exhibit different patterns, depending upon follicular developmental stage. Altogether, these results demonstrate that optimal follicle isolation depends on tissue thickness and ovarian region and that viable, good-quality preantral and antral follicles can be successfully isolated from equine ovarian tissue. LAY SUMMARY: Follicles are the functional units of the ovary that contain and support the egg. Depending on the presence of a fluid-filled cavity called an antrum, follicles are developmentally classified as preantral (no antrum) or antral (antrum present). In this study, small preantral and antral horse follicles were removed with needles from ovarian tissue sliced at different thicknesses and classified by their location and other characteristics. The health of the harvested follicles was then examined in several ways (cell survival, toxic by-product levels, and certain types of gene control) to evaluate how slice thickness during follicle removal affects these structures. This study reports, for the first time, that tissue slice thickness influences the removal of late preantral and early antral follicles in horses and that the location of these follicles depends on their developmental stage and quality, with these follicles showing distinct levels of gene control.