Semester of Graduation
Master of Science (MS)
Veterinary Clinical Sciences
A series of biochemical and physical modifications in the sperm during the transport in the female reproductive tract that make the sperm capable of fertilization are called capacitation. It is an important process since ejaculated sperm is not capable of fertilizing an oocyte before capacitation. The sperm modifications during the capacitation process include changes in membrane fluidity, tyrosine phosphorylation, calcium influx, pH increase and hyperpolarization, leading the sperm to achieve hypermotility and the acrosome reaction, which are also required for conventional in vitrofertilization (IVF). In vitro production of embryos leading to viable offspring has been successful in different mammalian species starting with the birth of a human baby in 1978 following an in vitro procedure that mimicked oocyte-sperm interactions, including capacitation of the sperm. In the horse, however, there is only one report of successful IVF and no other livebirths have been reported since then. Since it is possible to mature oocytes and culture equine embryos in vitro, fertilization itself still remains the main limiting factor of IVF in that species. Reports of equine IVF have shown poor results and even when fertilization was achieved, only a small number of embryos cleaved and/or was transferred. While in cattle heparin is an important factor for inducing sperm capacitation in vitro, in the horse, similar factors are still not characterized, therefore no acceptable rates of fertilization are attained. Multiple strategies and different compounds have been tested with limited success over the years, and it is difficult to reliably assess in vitro capacitation of equine sperm, especially due to the lack of slaughterhouse-derived oocytes for research, which also contributes to the slow progress in the area. The investigations conducted in my thesis research were inspired by recent reports in humans and mice linking protein lysine acetylation and sperm capacitation. Accordingly, an initial study was designed to first detect lysine acetylation in stallion sperm as a proof of principle, to test if lysine acetylation would increase during sperm incubation in vitro, and to further test if inducing a hyperacetylated state in the sperm would consequently induce an increase of capacitation- like changes such as hypermotility, increase of tyrosine phosphorylation and increase in acrosome reacted cells. For that, two deacetylase inhibitors (iDAC’s), namely trichostatin A and nicotinamide, were used in different concentrations and the sperm in vitro capacitation was evaluated by validated assessment protocols. Using computer-assisted semen analysis (CASA) using sperm from two fertile stallions, no hyperactivated motility was attained in the groups treated with capacitating, non-capacitating and different doses of iDAC’s, in both experiments. Total and progressive motility and viability were not significantly affected by the iDAC’s, but, on the other hand, the parameters were decreased in the capacitation group containing bicarbonate (HCO3), calcium and bovine serum albumin (BSA). No differences in acetylation were seen among groups or after treatment with iDAC’s. Only sperm in the capacitation group presented differences in other capacitation-like parameters such as higher tyrosine phosphorylation and acrosome reaction. The treatment with iDAC’s in turn, only increased the percentage of acrosome reacted sperm when the highest dose was used. In conclusion, capacitation events and equine sperm physiology still need to be further studied to understand the role of sperm acetylation. Our experiment showed that it is possible to detect acetylated proteins in equine sperm and induction of a hyperactivated state in the sperm might require higher doses of iDAC’s, different incubation times and different media pH modifications to achieve similar results previously found in murine species, where hyperacetylation induced capacitation-like changes in the sperm. Nevertheless, the use of equine oocytes or efficient heterologous fertilization protocols where sperm-oocyte interaction is possible, might help to understand why conventional IVF in horses does not work yet.
de Aguiar, Luis H., "Potential Role of Acetylation on Equine Sperm Capacitation" (2021). LSU Master's Theses. 5386.
Pinto, Carlos R.F.
Available for download on Monday, July 01, 2024