Effects of heat stress on oocyte quality and embryo viability in swine
Heat stress during oocyte maturation causes well-defined changes in the oocyte cytoplasm that leads to reduced developmental potential. Previous work has been done that supports this hypothesis, but the underlying reasons are still not fully understood. Our objective is to determine these underlying mechanisms that contribute to this reduction in overall development potential of the oocytes and the embryos. Oocytes underwent in vitro maturation (IVM) for 40-44 hours in one of two environments: 39°C (control), or 41°C (heat stress). The oocytes underwent multiple experiments: stage of meiosis after maturation, endoplasmic reticulum (ER) size, total amount of Ca2+ stored in the ER, pattern of Ca2+ signal following in vitro fertilization (IVF), and embryo development after IVF. After 40-44 hours of IVM, oocytes were stained and their chromatin configuration was recorded. Heat-stressed oocytes showed a significant decrease in the ability to reach metaphase II of meiosis compared to the control group. Heat stressed oocytes also lowered the percentage of embryos developing to the blastocyst stage. Mobilized Ca2+ from the ER affects fertilization and subsequent embryo development. Administration of ionomycin caused oocytes to, in Ca2+-free medium, released Ca2+ from the intracellular stores. The average amplitude of the Ca2+ peaks was significantly higher compared to that of the heat stressed group. However time of release and time to maximum peak did not seem to have a discernable affect between the groups. Lower Ca2+ store content could be caused by one of two events: reduction in ER size or an alteration in ER function. Literature suggests that the ER should emulate a spider web-type structure contained throughout the entire cytoplasm. In some cases, heat stress oocytes exhibited an incomplete reorganization of the ER as compared to the control group. Fertilization is dependent on the phospholipase C zeta (PLC ζ) content of the sperm to hydrolyze and cleave PIP2 into DAG and IP 3 to cause a release of Ca2+ from the ER. The released Ca2+ triggers a cascade of events that ultimately lead to the activation of the oocyte and subsequent embryo development. Oocyte lipid composition is important for the generation of energy substrates as well as phospholipid second messengers. Lipid analysis experiments did not show a significant difference in fatty acid make up. High amounts of triglycerides are seen in each experiment performed in positive mode. Phospholipids and DAG are in very low concentrations prior to fertilization. The DESI technique may not be sensitive enough to obtain the data from both groups. An alteration in fatty acid content as well as phospholipids can have a detrimental effect on Ca2+ mobilization and entry. Ca2+ transients, after fertilization, are responsible for the lifting of the meiotic arrest and initiation of cell division. When we measured the changes in the intracellular Ca2+ concentration of the oocytes we found that heat stress during maturation caused marked alterations in the mean amplitude of Ca2+ transients during IVF, while frequency of the oscillations remained the same. We believe that the observed changes described above are responsible, at least in part, for the reduced developmental potential of the heat stressed oocytes.
Machaty, Purdue University.
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