CALCIUM SIGNALING IN PIG OOCYTES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0233273
• Project Start Date: Oct 1, 2013
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Animal Sciences

Non Technical Summary
Accomplishing the objectives of this project is expected to provide the foundation required to significantly improve the efficiency of oocyte activation methods. Such methods are essential parts of assisted reproductive technologies such as in vitro fertilization or somatic cell nuclear transfer and thus have important agricultural implications. The sperm activates the oocyte and triggers embryo development by inducing repetitive increases in the oocyte's free calcium concentration. Repetitive calcium signals have physiological advantages over static calcium increases and have long-term effects on subsequent development. The repetitive behavior provides a means to deliver prolonged calcium signals to targets without the deleterious effects of sustained calcium elevations. The amplitude, frequency and duration of the sperm-induced calcium signals encode crucial information and have a profound effect on peri-implantation development in addition to effects on the immediate events of oocyte activation. Despite its significance, the complete mechanism of the fertilization calcium signal has never been characterized. It is well accepted that proper activation of the oocyte's developmental program is critical for the success of a number of assisted reproductive technologies, nuclear transfer, and fertilization under normal physiological conditions. Cloning by nuclear transfer of genetically modified cells is the only method on hand for making pigs with specific genetic modifications. Genetically modified animals would have vast potentials, the benefits they offer ranges from increased growth and improved product quality to enhanced disease resistance. However, our inability to properly activate the oocyte reconstructed during nuclear transfer is one of the reasons for the extreme inefficiency of nuclear transfer procedures. Because we don't know the underlying mechanism that mediates calcium signaling in fertilized oocytes we are not able to artificially induce the repetitive signals. The available parthenogenetic activation methods generate calcium changes that do not faithfully recapitulate those occurring after fertilization and this results in poor embryo development. Increasing our knowledge of how the sperm triggers the oscillatory calcium signals will enhance our abilities to more precisely control the process of signaling during embryo production in the laboratory.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3599	1030	35%
301	3599	1040	15%
303	3599	1030	35%
303	3599	1040	15%

Knowledge Area
303 - Genetic Improvement of Animals; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology;

Keywords

oocyte

fertilization

activation

calcium signal

calcium influx

calcium channel

pig

Goals / Objectives
A better understanding of the cellular and molecular mechanisms that underlie fertilization is key to improve the efficiency of assisted reproductive technologies and hence, to enhance productivity of animal agriculture. The present project focuses on calcium signaling that is responsible to induce oocyte activation and stimulate embryo development. The fertilizing sperm activates the oocyte's developmental program by triggering repetitive calcium rises in the oocyte's cytoplasm. Each elevation in the intracellular calcium concentration is followed by a calcium influx across the oocyte's plasma membrane. Although the entry of extracellular calcium is essential for proper development of the resulting embryo, little is known about the mechanism that mediates the influx and its exact role in oocyte activation is also unclear. The objectives of this project are twofold. 1. The molecular identity of the components of the entry pathway will be investigated. We are going to identify the molecules that are responsible for the generation of calcium entry across the plasma membrane. 2. The role of the transmembrane calcium current will be determined. Currently it is not known whether the calcium influx serves simply to replenish the calcium stores or whether it is also responsible for the activation of critical signaling pathways required for complete oocyte activation. Defining the mechanism and functions of calcium entry will deepen our knowledge about oocyte physiology and may also help improving the efficiency of a number of assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer.

Project Methods
The expression of known calcium influx channels will be downregulated in pig oocytes. The oocytes will then be inseminated and changes in the intracellular free calcium concentration monitored. Our hypothesis is that calcium entry channels are essential for periodical re-filling of the calcium stores during fertilization. If the calcium entry channels that function during fertilization are built of the protein that had been dowregulated in the oocytes, then fertilization would not be followed by calcium oscillations. These oocytes will show only the initial calcium rise as a result of mobilization of calcium stored in the endoplasmic reticulum. To further investigate the mechanism that mediates calcium entry, protein fragments that interfere with the interaction between components of critical calcium influx pathways will be expressed in pig oocytes. A possible mechanism to generate calcium influx is store-operated calcium entry mediated by an interaction between STIM1 and Orai1 proteins. The N-terminal domain of Orai1 (amino acid residues 1-90; Orai1-NT) and the C-terminal domain of STIM1 (amino acid residues 238-685; STIM1-CT) will be expressed in the oocytes. The oocytes will then be fertilized and changes in the intracellular free calcium concentration monitored. The absence of repetitive calcium transients will be an indicator that calcium influx at fertilization requires an interaction between STIM1 and Orai1. Results in our laboratory demonstrated that oocytes lacking STIM1 or Orai1 showed only a single calcium rise at fertilization. It seems to indicate that these proteins are required during fertilization. However, it may also suggest that STIM1 and Orai1 are needed prior to fertilization, during maturation, when the calcium stores in the oocyte undergo reorganization. To test this possibility STIM1 or Orai1 levels will be downregulated in immature pig oocytes. Following maturation the calcium stores in the oocytes will be depleted and the amount of calcium released will be measured. A reduced level of calcium released from the intracellular stores would indicate that STIM1 or Orai1 are required during maturation to provide calcium influx for loading the calcium stores. Finally, recent experimental data in the mouse seem to indicate that calcium influx during fertilization is essential not only to replenish the intracellular calcium stores but also, to induce complete activation of the oocyte. To investigate the importance of repetitive calcium transients versus calcium influx in porcine oocyte activation, both influx and efflux of calcium will be blocked using appropriate inhibitors. Fertilization under such conditions results in repetitive calcium transients without calcium influx. In another set of experiments the effect of calcium influx in the absence of repetitive calcium oscillations will be tested using calcium chelators. This treatment is expected to lead to a single sperm-induced calcium rise followed by calcium influx. The oocytes will be fertilized and early and late oocyte activation events will be monitored to determine the role and significance of calcium influx during fertilization.

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:General scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project providid training opportunities for 2 undergraduate students and 1graduate student. The students became familiar with techniques related to cell culture, in vitro oocyte maturation, in vitro fertilization, embryo culture, cRNA production and fluorescence resonance energy transfer. How have the results been disseminated to communities of interest?The results have been published in journal articles and conference proceedings. What do you plan to do during the next reporting period to accomplish the goals?We are designing new experiments to better understand the role of magnesium and zinc during fertilizationof pig eggs.

Impacts
What was accomplished under these goals? We demonstrated previously that store-operated calcium entry across the egg plasma membrane is essential fornormal fertilization. We showed that STIM1 is a protein located in the membrane of the intracellular stores known as theendoplasmic reticulum, and it is able to detect calcium levels in the stores. We also showed that the calcium influx channelsare made of Orai1 proteins. Using Fluorescence Resonance Energy Transfer (FRET) we showed that repetitive FRETchanges occur in the eggs following sperm-egg fusion, indicating thatfertilization is associated with repetitive interaction between STIM1 and Orai1. Recently we applied appropriate inhibitors tostrengthen the notion that STIM1 and Orai1 proteins are essential to sustain the fertilization calcium signal in pig oocytes.Inaddition, we demonstrated the role of magnesium in the regulation of the calcium signal. In a series of experiments we showed that magnesium blocks calcium entry, probably by inhibiting the kinase domain of the TRPM channel, which in turnblocks store-operated calcium entry. This supports our major hypothesis that store-operated calcium entry mediated bySTIM1 and Orai1 proteins is essential to sustain the sperm-induced calcium signal during fertilization in pig eggs. I addition, we also demonstrated the role of zinc in eggactivation. We showed that pig eggs can artificially activated using zinc chelators; this shows that changes in theintracellular zinc concentration in aggs plays a role in stimulating embryo development.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang L, Chao C-H, Jaeger LA, Bali Papp A, Machaty Z. 2018. Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1. Biology of Reproduction (doi: 10.1093/biolre/ioy016).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Machaty Z. 2018. The use of pigs in future biotechnology. (Hungarian). The Swine (in press).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Li Z, Ehmke N, Machaty Z, Nolte D. 2018. A potential non-invasive approach to evaluating blastocyst quality using biodynamic imaging. Proc. SPIE 10472, Diseases in the Breast and Reproductive System IV, 100631G.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zartman M, Ehmke N, Machaty Z. 2018. In vitro maturation of porcine oocytes in different media. Proc. Purdue Undergraduate Research Conference. West Lafayette, Indiana.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Uh K, Ryu J, Zhang Lu, Errington J, Machaty Z, Lee K. 2018. Artificial oocyte activation using novel Zn2+ chelators in pigs. Proc 24th Meeting of the Society of Reproductive Biology, Szekesfehervar, Hungary.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:General scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project providid training opportunities for 2 undergraduate and 1graduate students. The students became familiar withtechniques related to cell culture, in vitro oocyte maturation, in vitro fertilization, embryo culture, cRNA production and FRET. How have the results been disseminated to communities of interest?The results have been published in conference proceedings and book chapters. What do you plan to do during the next reporting period to accomplish the goals?We are designing new experiments to better understand the role of magnesium in the regulation of calcium signaling during fertilization of pig oocytes.

Impacts
What was accomplished under these goals? We demonstrated previouslythat store-operated calcium entry across the egg plasma membrane is essential for normalfertilization. We showed that STIM1 is a protein located in the membrane of the intracellular stores known as the endoplasmicreticulum, and it is able to detect calcium levels in the stores. We also showed that the calcium influx channels are made ofOrai1 proteins. Using Fluorescence ResonanceEnergy Transfer (FRET) we showed that repetitive FRET changes occur in the eggs following sperm-egg fusion, indicating that fertilization is associated with repetitive interaction between STIM1 and Orai1. Recently we applied appropriate inhibitors to strengthen the notion that STIM1 and Orai1 proteins are essential to sustain the fertilization calcium signal in pig oocytes. Inaddition, we demonstrated the role of magnesium in the regulation of the calcium signal. In a series of experiments we showed that magnesium blockscalcium entry, probably by inhibiting the kinase domain of the TRPM channel, which in turn blocks store-operated calcium entry.This supports our major hypothesis that store-operatedcalcium entry mediated by STIM1 and Orai1 proteins is essential to sustain the sperm-induced calcium signal during fertilizationin pig eggs.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Machaty Z. 2017. Oocyte activation at fertilization. Proc Society of Reproductive Biology, Cegled, Hungary, p. 10.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Machaty Z, Miller AR, Zhang L. 2017. Egg activation at fertilization. In: Vertebrate development. (Eds.: F. Pelegri, M. Danilchik, A. Sutherland). Adv. Exp. Med. Biol., Vol. 953. pp. 1-47. Springer, Cham, Switzerland.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Machaty Z, Wang C, Lee K, Zhang L. 2017. Fertility: Store-operated Ca2+ entry in germ cells: role in egg activation. In: Store-operated Ca2+ entry (SOCE) pathways, Second edition; (Eds.: K. Groschner, W.F. Graier, C. Romanin). Pp. 577-593. Springer-Verlag/Wien, Austria.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhang L, Machaty Z. 2017. The effect of magnesium on the sperm-induced calcium signal in porcine oocytes. Proc Society for the Study of Reproduction, Washington, DC, p. 109.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:General scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project providid training opportunities for 2 undergraduate and 2 graduate students. The students became familiar with techniques related to cell culture, in vitro oocyte maturation, embryo incubation in vitro, cRNA production and FRET. How have the results been disseminated to communities of interest?Some of the results have been published in peer-reviewed journals and conference proceedings. Another manuscript is currently under preparation. What do you plan to do during the next reporting period to accomplish the goals?We are planning to investigate whether the Orai1 channel can transport magnesium intothe egg cytoplasm. In some species, the in vitro fertilization medium also contains magnesium. If the Orai1 channel delivers not only calcium but magnesium ions as well, that may affect the efficiency of egg activation at fertilization.

Impacts
What was accomplished under these goals? We demonstrated earlier that store-operated calcium entry across the egg plasma membrane is essential for normal fertilization. We showed that STIM1 is a protein located in the membrane of the intracellular stores known as the endoplasmic reticulum, and it is able to detect calcium levels in the stores. We also showed that the calcium influx channels aremade of Orai1 proteins. Recently we investigated the interaction between the two proteins using the method of Forster Resonance Energy Transfer (FRET). We expressed STIM1 and Orai1 proteins tagged with appropriate light-sensitive molecules (fluorophores) in porcine eggs. The eggs were then fertilized in vitro and changes in FRET were monitored by means of an imaging system.We were able to detectrepetitive FRET changes in theeggs following sperm-egg fusion, indicating that fertilization is associated with repetitive interaction between STIM1 and Orai1. This supports our hypothesis that store-operated calcium entry mediated by STIM1 and Orai1 proteins is activated at fertilizationin pig eggs.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Machaty Z. 2016. Signal transduction in mammalian oocytes during fertilization. Cell Tissue Res 363:169-183.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Wang C, Machaty Z. 2016. Characterization of the first sperm-induced calcium transient in pig oocytes. Reprod Fertil Dev 28:225.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Silva PV, Zhang L, Jaeger LA, Machaty Z. 2016. Effects of ORAI1 down-regulation on oocyte maturation in pigs. Proc 18th International Congress on Animal Reproduction. Tours, France, p.280.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhang L, Chao C-H, Jaeger LA, Machaty Z. 2016. Repetitive interactions between STIM1 and ORAI1 at fertilization in pig oocytes. Proc 18th International Congress on Animal Reproduction. Tours, France, p. 50.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:General scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for an undergraduate and a graduate student. The students learnt laboratory techniques related to cell culture, in vitro maturation of pig oocytes, embryo culture, in vitro transcription of DNA, and calcium imaging. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?We are planning to downregulate the level of STIM1 and Orai1 in pig oocytes. They will be fertilized and the resulting embryos cultured in vitro. We will then evaluate the embryos to determine their viability. It is expected that the embryos that were generated from STIM1- or Orai1-deficient oocytes are less viable, and express genes that are required for embryo development in a faulty manner.

Impacts
What was accomplished under these goals? Previously we have identified store-operated calcium entry (SOCE) as a mechanism essential to sustain the long-lasting calcium signal during fertiliztion in pig oocytes. We have further investigated the role of this calcium entry mechanism during the oocyte-embryo transition. We found that 10 μM gadolinium, which is known to inhibit SOCE, blocked calcium entry that was triggered by thapsigargin-induced store depletion and also caused an abrupt cessation of the fertilization calcium signal. In a similar manner 3,5-bis(trifluoromethyl)pyrazole 2 (20 μM), and tetrapandin-2 (10 μM), potent SOCE inhibitors, also blocked thapsigargin-stimulated calcium entry and disrupted the calcium oscillations after sperm-egg fusion. The downregulation of Stim1 or Orai1, the proteins that mediate SOCE, in the eggs did not alter the calcium content of the intracellular stores, whereas co-overexpression of these proteins led to the generation of irregular calcium transients after fertilization that stopped prematurely. We also found that thapsigargin completely emptied the endoplasmic reticulum, and that the series of calcium transients stopped abruptly after the addition of thapsigargin to the fertilized eggs, indicating that the proper reloading of the intracellular stores is a prerequisite for the maintenance of the calcium oscillations. These data strengthen our previous findings that in porcine eggs SOCE is a major signaling cascade that is responsible for sustaining the repetitive calcium signal at fertilization.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang C, Zhang L, Jaeger LA, Machaty Z.Store-Operated Ca2+ Entry Sustains the Fertilization Ca2+ Signal in Pig Eggs.Biol Reprod. 2015 Jul;93(1):25. doi: 10.1095/biolreprod.114.126151.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Machaty Z.Signal transduction in mammalian oocytes during fertilization. Cell Tissue Res. 2015 Oct 9. [Epub ahead of print]
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lee K, Davis A, Zhang L, Ryu J, Spate LD, Park KW, Samuel MS, Walters EM, Murphy CN, Machaty Z, Prather RS. Pig oocyte activation using a Zn(2+) chelator, TPEN. Theriogenology. 2015 Oct 1;84(6):1024-32.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: General scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training opportunities for an undergraduate and a graduate student. The students learnt laboratory techniques related to cell culture, in vitro maturation of pig oocytes, embryo culture and calcium imaging. How have the results been disseminated to communities of interest? The results have been presented at an international scientific conference and published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We found that in pig eggs an influx of calcium ions is essential to maintain the repetitive calcium signal that activates the egg during fertilization. The calcium entry is mediated by two proteins, STIM1 and Orai1. STIM1 senses calcium content in the intracellular calcium stores and after store depletion it transduces the empty signal to the plasma membrane. Orai1 is a calcium entry channel in the plasma mebrane and when activated by STIM1 it lets calcium into the cell. The mechanism is termed store-operated calcium enry and it seems to be essential in pig eggs for the generation of the sustained calcium signal at fertilization and also, for the stimulation of normal embryo development. Experiments were designed to characterize the calcium entry pathway that operates at fertilization. We found that low concentrations of gadolinium, which is known to inhibit store-operated calcium entry, blocked calcium entry that was triggered by store depletion and it also caused an abrupt cessation of the fertilization calcium signal. At the same time 2-aminoethyldiphenyl borate (2-APB), a store-operated calcium entry modulator, at low micromolar concentrations potentiated store-operated calcium entry and disrupted the calcium oscillations after sperm-egg fusion. Co-overexpression of these proteins led to the generation of irregular calcium transients after fertilization that stopped prematurely. We also found that pig eggs do not have a thapsigargin-insensitive calcium pool since ionomycin or inositol 1,4,5-trisphosphate was unable to mobilize additional calcium following store depletion induced by thapsigargin and the series of calcium transients stopped abruptly after the addition of thapsigargin to the fertilized eggs. These data strengthen our previous findings that in porcine eggs store-operated calcium entry is a major signaling cascade that is responsible for sustaining the repetitive calcium signal at fertilization.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Lee, K., Wang, C., Spate, L., Murphy, C.N., Prather, R.S., Machaty, Z. 2014. Gynogenetic activation of porcine oocytes. Cell. Reprogram. 16:121-129.
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Wang, C., Zhang, L., Jaeger, L.A., Machaty, Z. 2014. Store-operated Ca2+ entry sustains the fertilization Ca2+ signal in pig eggs. Biol. Reprod.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Pirro, V., Oliveri, P., Ferreira, C.R., Gonz�lez-Serrano, A.F., Machaty, Z., Cooks, R.G. 2014. Lipid characterization of individual porcine oocytes by dual mode DESI-MS and date fusion. Anal. Chim. Acta. 848:51-60.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Machaty, Z. 2013. Calcium influx during fertilization in mammalian oocytes. J. Vet. Sci. Technol. 4:36.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Machaty, Z. 2013. Signal transduction during fertilization in pig oocytes with special regard on gene preservation. 2nd Fatty Pig Science and Utilization International Conference, Herceghalom, Hungary, p.13.