Fluorescent enzyme-based biosensor for sensitive analysis of DNA damage in cryopreserved sperm.

The freeze-thaw process can induce irreversible structural and functional changes in human sperm, particularly sperm DNA damage. Selecting a more accurate and sensitive detection method for evaluating sperm DNA integrity is crucial. To accurately assess sperm DNA integrity following the freeze-thaw process and significantly improve the clinical and scientific utilization of cryopreserved sperm. In this study, we utilized a novel fluorescent biosensor, assisted by terminal deoxynucleotidyl transferase (TdT) and Endonuclease IV, to detect DNA breakpoints during sperm cryopreservation. We evaluated the biosensor's performance by comparing it with the conventional DNA fragmentation index (DFI) measured using sperm chromatin structure analysis (SCSA). The cryopreserved group exhibited a significantly higher sperm DFI compared to the fresh group. No significant difference was observed between the antioxidant group and the cryopreserved group. However, the new method revealed a significant reduction in the number of DNA breakpoints in the antioxidant group compared to the cryopreserved group. The novel biosensor demonstrated superior accuracy and effectiveness in assessing sperm DNA integrity during cryopreservation compared to the conventional SCSA method. We believe that the biosensor holds significant potential for widespread use in the field of reproductive medicine.

The role of miR-199a-3p in inhibiting the proliferation of spermatogonial stem cells under heat stress.

MicroRNAs (miRNAs) play a crucial role in regulating various physiological processes, including cell differentiation, proliferation, and apoptosis. However, their specific functions in response to heat stress are not fully understood. This study aimed to investigate the regulatory effects of miR-199a-3p on the proliferation of heat stress-treated spermatogonial stem cells (SSCs). SSCs were isolated from mouse testes and cultured in vitro to identify marker molecules. Lentiviruses carrying miR-199a-3p-over, miR-199a-3p-inhibit, and ID4-over constructs were generated for stable transfection. Luciferase assay was employed to confirm the targeting relationship between miR-199a-3p and ID4. An in vitro SSCs heat stress model was established, and the miR-199a-3p-inhibit and ID4-over groups were included. Cellular proliferation was assessed using CCK-8, EdU, and cell cycle analysis methods after heat stress. Expression levels of miR-199a-3p and ID4 were evaluated by western blotting and qRT-PCR. The results demonstrated that miR-199a-3p-over inhibited SSCs proliferation, while ID4-over promoted an increase in SSCs number. Luciferase assay confirmed the regulatory effect of miR-199a-3p on ID4 expression. Moreover, after heat stress treatment, miR-199a-3p-inhibit and ID4-over enhanced SSCs proliferation compared to the control group. These findings suggest that miR-199a-3p modulates SSCs proliferation by targeting ID4, especially under heat stress conditions.

Localization of nerve entry points as targets to block spasticity of the deep posterior compartment muscles of the leg.

To identify the optimal body surface puncture locations and the depths of nerve entry points (NEPs) in the deep posterior compartment muscles of the leg, 60 lower limbs of thirty adult cadavers were dissected in prone position. A curved line on the skin surface joining the lateral to the medial epicondyles of the femur was taken as a horizontal reference line (H). Another curved line joining the lateral epicondyle of the femur to the lateral malleolus was designated the longitudinal reference line (L). Following dissection, the NEPs were labeled with barium sulfate and then subjected to spiral computed tomography scanning. The projection point of the NEP on the posterior skin surface of the leg was designated P, and the projection in the opposite direction across the transverse plane was designated P'. The intersections of P on H and L were identified as PH and PL , and their positions and the depth of the NEP on PP' were measured using the Syngo system and expressed as percentages of H, L, and PP'. The PH points of the tibial posterior, flexor hallucis longus and flexor digitorum longus muscles were located at 38.10, 46.20, and 55.21% of H, respectively. The PL points were located at 25.35, 41.30, and 45.39% of L, respectively. The depths of the NEPs were 49.11, 54.64, and 55.95% of PP', respectively. The accurate location of these NEPs should improve the efficacy and efficiency of chemical neurolysis for treating spasticity of the deep posterior compartment muscles of the leg. Clin. Anat. 30:855-860, 2017. © 2017 Wiley Periodicals, Inc.