Date of Award

Fall 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Animal Science

First Advisor

Zoltan Machaty

Committee Chair

Zoltan Machaty

Committee Member 1

Ryan Cabot

Committee Member 2

Paul Collodi

Committee Member 3

Laurie Jaeger

Committee Member 4

Shihuan Kuang

Abstract

In a variety of mammalian cells, the depletion of intracellular Ca2+ stores leads to Ca2+ influx across the plasma membrane though a process known as store-operated Ca2+ entry. It not only plays a critical role in the replenishment of the Ca2+ stores but it is also responsible for the maintenance of long-lasting high intracellular Ca2+ levels and sustaining repetitive Ca2+ oscillations. During fertilization a Ca2+ signal, which can take the form of a single or multiple Ca2+ elevations (depending on the species) is the universal trigger for the egg-to-embryo transition. For sustaining the train of Ca2+ spikes an influx of Ca2+ across the plasma membrane is an absolute pre-requisite. However, despite its significance little is known about the mechanism that mediates Ca2+ influx at fertilization. Previously we have shown that STIM1, a key component of the store-operated calcium entry pathway plays an important role in regulating the oscillatory Ca2+ signal during porcine fertilization.

In the present study we dissected the mechanism that maintains the repetitive fertilization Ca2+ signal. Using gadolinium (Gd3+) we specifically inhibited Ca2+ channels gated by the filling status of the intracellular Ca2+ stores in order to better understand this critical signaling cascade. We found that in control eggs store depletion in Ca2+ -free medium followed by Ca2+ add-back triggered an elevation in the cytosolic Ca2+ levels indicating store-operated Ca2+ entry. This Ca2+ influx was completely blocked in gadolinium pre-treated oocytes. Furthermore, the sperm-induced Ca2+ oscillations were also abolished by the addition of gadolinium and 2-APB, a modulator of store-operated Ca2+ entry also disrupted the fertilization Ca2+ signal. These results indicate that the sperm-induced Ca2+ oscillations in pig eggs are maintained via a Ca2+ influx generated by the depletion of the intracellular Ca2+ stores.

In order to better understand the role of store-operated Ca2+ entry during fertilization, we studied Orai1, the proposed channel component of this signaling pathway. Through RT-PCR we demonstrated the presence of Orai1 transcripts in pig eggs; the results of Western blot analysis further confirmed that Orai1 is expressed in pig eggs. In addition, we cloned the entire porcine Orai1 coding sequence and tagged it with the enhanced green fluorescence protein (EGFP). Confocal imaging after microinjection of the mRNA of this EGFP-Orai1 fusion protein indicated that EGFP-Orai1 localized primarily in the cortical region of the eggs. By using antibodies raised against Orai1 we further verified that Orai1 was located mainly at the plasma membrane. Next, we found that Orai1 expression decreased significantly during the course of oocyte maturation, whereas STIM1 expression remained unchanged. We also noted that during maturation the oocytes gain the ability to generate a large Ca2+ influx after store depletion which indicates the development of the store-operated Ca2+ entry pathway that is required for fertilization. We further investigated the role of Orai1 in generating Ca2+ signals during fertilization. Microinjection of siRNA against Orai1 abolished the repetitive sperm-induced Ca2+ transients and only a single Ca2+ elevation with a markedly smaller amplitude was generated. The embryos that developed from these eggs after in vitro fertilization showed significantly lower developmental potential. When Orai1 was overexpressed in the eggs by microinjecting EGFP-Orai1 mRNA, the elevated Orai1 level disrupted the normal Ca2+ oscillations and co-overexpression of STIM1 and Orai1 also had a negative effect on the Ca2+ oscillations leading to highly elevated Ca2+ levels.

In summary, we demonstrated that store-operated Ca2+ entry is the major mechanism responsible for mediating Ca2+ influx during fertilization in pig eggs. Together with STIM1, the other major component of the pathway, Orai1 plays an important role in sustaining the oscillatory Ca2+ signal and its proper function during fertilization is essential for subsequent embryo development. Further studies are under way to elucidate the communication between STIM1 and Orai1 to better understand the regulatory mechanisms that operate during fertilization.

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