Objective:To establish and evaluate a follicle isolation method, combination of enzymatically digestion with strainer filtration, for synchronously collecting mouse follicles across different developmental stages.Methods:Pre-cutted ovarian tissue blocks were digested with enzymes mixed by Collagenase I and DNase I, and then filtered through three different pore size cell strainers to synchronously separate the follicles at developmental stages. The follicles obtained by turning over and washing the 100 μm strainer were recorded as group A (>100 μm), as same as the follicles obtained by turning over and washing the 40 μm strainer were recorded as group B (40-100 μm), and the follicles obtained by turning over and washing the 20 μm strainer were recorded as group C (20-40 μm). The quantity, morphology, viability and developmental potency were examined among these harvested follicles.Results:In terms of quantity, follicles in group C accounted for most, followed by follicles in group A, and the follicles in group B were the least. Morphologically, the basal membrane integrity rate of follicles in group C was higher than that of groups B and A ( P<0.001 and P<0.001, respectively). As for viability, the survival rate of follicles in group A was 70.59% (120/170), which was higher than that in group B (51.79%, 58/112) and group C (35.90%, 28/78) ( P=0.001 6 and P<0.001, respectively). In terms of developmental potency, after 96 h of in vitro culture, the diameter of follicles in group A increased from (113.64±9.57) μm to (150.95±45.90) μm ( P=0.002 4), and the diameter of follicles in group B increased from (88.12±9.12) μm to (120.61±18.00) μm ( P<0.001). In group C, monolayer-layer granulosa cells were attached to the follicles within 24 h of culture, and the connection structure between oocytes and granulosa cells was lost. The oocytes were completely exposed and failed to be cultured successfully. Conclusion:Combining enzymatically digestion with multiple strainers filtration is a rapid and effective method for follicle collecting, which is capable to isolate different developmental stage mouse follicles synchronously with morphological integrity, favorable viability and good developmental potency.

Objective:To establish and evaluate a follicle isolation method, combination of enzymatically digestion with strainer filtration, for synchronously collecting mouse follicles across different developmental stages.Methods:Pre-cutted ovarian tissue blocks were digested with enzymes mixed by Collagenase I and DNase I, and then filtered through three different pore size cell strainers to synchronously separate the follicles at developmental stages. The follicles obtained by turning over and washing the 100 μm strainer were recorded as group A (>100 μm), as same as the follicles obtained by turning over and washing the 40 μm strainer were recorded as group B (40-100 μm), and the follicles obtained by turning over and washing the 20 μm strainer were recorded as group C (20-40 μm). The quantity, morphology, viability and developmental potency were examined among these harvested follicles.Results:In terms of quantity, follicles in group C accounted for most, followed by follicles in group A, and the follicles in group B were the least. Morphologically, the basal membrane integrity rate of follicles in group C was higher than that of groups B and A ( P<0.001 and P<0.001, respectively). As for viability, the survival rate of follicles in group A was 70.59% (120/170), which was higher than that in group B (51.79%, 58/112) and group C (35.90%, 28/78) ( P=0.001 6 and P<0.001, respectively). In terms of developmental potency, after 96 h of in vitro culture, the diameter of follicles in group A increased from (113.64±9.57) μm to (150.95±45.90) μm ( P=0.002 4), and the diameter of follicles in group B increased from (88.12±9.12) μm to (120.61±18.00) μm ( P<0.001). In group C, monolayer-layer granulosa cells were attached to the follicles within 24 h of culture, and the connection structure between oocytes and granulosa cells was lost. The oocytes were completely exposed and failed to be cultured successfully. Conclusion:Combining enzymatically digestion with multiple strainers filtration is a rapid and effective method for follicle collecting, which is capable to isolate different developmental stage mouse follicles synchronously with morphological integrity, favorable viability and good developmental potency.