Calcium Signalling in the Regulation of Flagellar Beating in Sperm

Cornell Univ - State: Awds Made Prior May 2010, Ithaca NY

Investigators

Abstract

Calcium Signalling in the Regulation of Flagellar Beating in Sperm In mammals, fertilization takes place deep within the female reproductive tract, in the ampulla of the oviduct. When sperm are recovered from the ampulla and observed swimming on microscope slides, they swim vigorously in circles. This swimming pattern is termed hyperactivation. In the environment of the oviduct, hyperactivation is not so useless as it might seem; in fact, it assists sperm in reaching the oocyte and in penetrating the cumulus matrix and zona pellucida. Hyperactivation is switched on by an increase in Ca 2+ at the flagellar axoneme in the core of the flagellum. The goal of this project is to determine how Ca 2+ reaches the axoneme to switch on hyperactivation. Pharmacological agents that induce release of Ca 2+ from intracellular stores (thapsigargin, thimerosal, and caffeine) were found to initiate hyperactivation in bull sperm. There are two families of Ca 2+ stores in cells, the inositol 1,4,5-trisphosphate receptor stores (IP3 R) and the ryanodine receptor stores (RyR). Antibodies against IP3 R revealed the presence of a store in the flagellum. This store may be the redundant nuclear envelope (RNE), an organelle located at the base of the flagellum. The RNE arises during spermiogenesis, when condensation of the nucleus of the spermatid leaves behind excess nuclear envelope. Rather than being discarded, as are all other superfluous cytoplasm and organelles, the RNE is retained and packaged neatly as a scroll of membranes at the base of the flagellum. It is proposed that the RNE is retained to serve as a Ca 2+ store. In addition to providing Ca 2+ to the base of the axoneme, the RNE could release Ca 2+ to mitochondria to stimulate ATP production. The RNE is quite closely associated with mitochondria at the base of the flagellum. These mitochondria are terminal members of the tightly-wrapped helix of mitochondria that surrounds the axoneme in the flagellar midpiece. The helix forms a barrier in the midpiece between the plasma membrane and axoneme; therefore, the mitochondria are also in a position to regulate Ca 2+ in the axoneme. Given this unique organization of RNE and mitochondria with respect to the axoneme, the following 4 hypotheses were developed to test how these organelles play a role in initiating hyperactivation. Bull sperm (Bos taurus) will be used to test the hypotheses. Hypothesis 1: The intracellular store that provides Ca 2+ for hyperactivation is gated by both IP3 and ryanodine receptors. Our preliminary evidence implicates both types of receptors in releasing Ca 2+ to initiate hyperactivation. Highly specific agonists of the receptors will be used to confirm these findings. If the hypothesis is correct, it would mean that the amount of Ca 2+ released can occur at two or three levels, depending upon which receptor is activated: IP3 R alone, RyR alone, or the two in combination. Such a mechanism can control the intensity of hyperactivation, such as to increase flagellar bending during penetration of the zona pellucida. Hypothesis 2: The intracellular Ca 2+ store in the flagellum is the RNE. It is proposed that this unique structure, which has been ignored in the past, is responsible for providing Ca 2+ for hyperactivation. Localization of the IP3 R and/or RyR to the RNE should tell us whether this hypothesis is correct. Hypothesis 3: A Ca 2+ rise in mitochondria is involved in switching on hyperactivation. The picture of Ca 2+ signalling that has been emerging for other cells is that mitochondria pick up Ca 2+ released from intracellular stores and respond by increasing ATP production. ATP levels are doubled in hyperactivated guinea pig sperm. Therefore, experiments are proposed to test whether release of Ca 2+ from intracellular stores results in increased levels of Ca 2+ , NADH, and ATP in flagellar mitochondria. Hypothesis 4: Mitochondria regulate flagellar Ca 2+ and hyperactivation. The tight wrap of mitochondria around the flagellar axoneme separates it from the plasma membrane; therefore, mitochondria are in a position to affect Ca 2+ levels at the axoneme in the site where the flagellar bend develops. Blockers of mitochondrial release of Ca 2+ will be applied to sperm before using pharmacological initiators of hyperactivation to determine whether hyperactivation can be prevented. The testing of these 4 hypotheses should provide a picture of how Ca 2+ turns on this movement pattern that is essential to fertilization.