Semester of Graduation
Master of Science (MS)
This study assessed the effects both DTT and progesterone have on bovine spermatozoa to induce decondensation or the acrosome reaction in order to facilitate male pronuclear formation after ICSI. Sperm prepared by swim-up in a 5 mM concentration of DTT displayed time dependent morphological changes resulting in decondensation over a 6-hour period. An estimated 90% of treated sperm displayed early changes in morphology after the second hour of incubation. Sperm displaying partial decondensation of the nucleus was estimated as 42% by the 3rd hour and increased to 62% by the 5th hour of incubation. Fully decondensed sperm or sperm with completely dispersed DNA increased from 20% at hour 5 to 50% at hour 7. No such changes were seen in sperm not treated with DTT (p < 0.01). Progesterone was used to induce an acrosome reaction in sperm which were first capacitated with heparin (10 µg/mL) prior to treatment with progesterone (10 µM). The acrosome reacting population was measured using a conjugated FITC-PNA stain and observations using fluorescent microscopy and cytometric flow analysis. Sperm undergoing an acrosome reaction at hour 1 was 80.2% and increased to 89.3% after 2 hours. Sperm that completed the acrosome reaction increased from 49.8% at hour 1 and 62.5% at hour 2. Only 14.2% completed acrosome reactions in sperm not treated with progesterone (p < 0.05). Using these observations with DTT and progesterone treated sperm, ICSI was performed with both treatments to assess if rates of male pronuclei formation and successful fertilization could be improved. Injection of sperm treated with DTT resulted in 28.3% of embryos with 2 pronuclei (2PN) after activation and 16 hours of culture, compared to 6% in control injections (p < 0.001). ICSI with progesterone treated sperm also resulted in a higher number of 2PN embryos, with 38.1% compared to 10.1% in control injections (p < 0.001). These results show that both DTT and progesterone have effects on sperm which allow for higher rates of male pronuclear formation after ICSI by utilizing either the physical approach of DTT or the sperm’s physiological response to progesterone.
Gatenby, Lauren, "Sperm Decondensation and Male Pronuclear Formation in Bovine Intracytoplasmic Sperm Injection" (2020). LSU Master's Theses. 5179.