Periodic Reporting for period 1 - Egg-Cortex (Actomyosin driven force-generation at the egg surface during fertilization)
Berichtszeitraum: 2023-02-01 bis 2025-01-31
Infertility is a significant global health challenge, impacting 8 to 12% of couples of reproductive age. Traditionally, infertility research has heavily focused on sperm cells, with discoveries in sperm motility and activation propelling advancements in in vitro fertilization (IVF) techniques. However, this has led to a relative under-exploration of the egg's role in fertilization. Notably, while eggs are known to actively prevent polyspermy and facilitate cell-cell fusion, the intricate subcellular mechanisms underpinning these processes remain largely uncharted. This project is conceived in light of the critical knowledge gap in understanding the egg's active role in fertilization. Specifically, it seeks to elucidate the dynamic modulation of the egg actomyosin cortex, a crucial player during the fertilization process. Given the complex and rapid nature of fertilization, involving overlapping stages of gamete activation, adhesion, fusion, and sperm incorporation, previous research has been limited by the lack of suitable live-cell microscopy techniques.
To overcome these limitations, our project will employ an innovative methodological framework using eggs isolated from zebrafish. This model organism offers a unique advantage as its eggs can be activated independently of sperm interaction, allowing for detailed observation and analysis of the fertilization process. In total, we have set forth four interrelated objectives:
Objective I revolves around a detailed characterization of the actin cortex in zebrafish eggs. We plan to investigate how this cortex changes over time, both before and after the egg is activated. A special emphasis will be placed on understanding the variations in intracellular calcium levels and the process of Cortical Granule Exocytosis (CGE), as these are pivotal in egg activation and the prevention of polyspermy.
Objective II aims to shed light on the role of the egg actin cortex in the fusion of gametes. We seek to unravel how this cortex facilitates the process of sperm uptake, a vital step for successful fertilization. Understanding this interaction at a molecular level could offer new insights into the fertilization process.
In Objective III, we explore a novel concept – the oocyte surface as a mechanosensory interface. This objective is focused on understanding how the egg's surface reacts to physical forces during sperm interaction. Specifically, we are interested in how these interactions might alter the cortical stiffness, potentially making the egg more conducive to cell-cell fusion.
Finally, Objective IV involves a targeted interference approach to strategically disrupt actin structures within the egg. By doing so, we aim to gauge the effects of such disruptions on fertilization rates. This objective is particularly crucial as it will allow us to connect our findings back to the specific structures and processes identified in the previous objectives, offering a comprehensive understanding of their physiological significance.
The outcomes of this project are anticipated to significantly advance our understanding of the egg's role in fertilization, particularly regarding the function of the actin cortex.
To overcome these limitations, our project will employ an innovative methodological framework using eggs isolated from zebrafish. This model organism offers a unique advantage as its eggs can be activated independently of sperm interaction, allowing for detailed observation and analysis of the fertilization process. In total, we have set forth four interrelated objectives:
Objective I revolves around a detailed characterization of the actin cortex in zebrafish eggs. We plan to investigate how this cortex changes over time, both before and after the egg is activated. A special emphasis will be placed on understanding the variations in intracellular calcium levels and the process of Cortical Granule Exocytosis (CGE), as these are pivotal in egg activation and the prevention of polyspermy.
Objective II aims to shed light on the role of the egg actin cortex in the fusion of gametes. We seek to unravel how this cortex facilitates the process of sperm uptake, a vital step for successful fertilization. Understanding this interaction at a molecular level could offer new insights into the fertilization process.
In Objective III, we explore a novel concept – the oocyte surface as a mechanosensory interface. This objective is focused on understanding how the egg's surface reacts to physical forces during sperm interaction. Specifically, we are interested in how these interactions might alter the cortical stiffness, potentially making the egg more conducive to cell-cell fusion.
Finally, Objective IV involves a targeted interference approach to strategically disrupt actin structures within the egg. By doing so, we aim to gauge the effects of such disruptions on fertilization rates. This objective is particularly crucial as it will allow us to connect our findings back to the specific structures and processes identified in the previous objectives, offering a comprehensive understanding of their physiological significance.
The outcomes of this project are anticipated to significantly advance our understanding of the egg's role in fertilization, particularly regarding the function of the actin cortex.
My project, planned for a 24-month period, was structured into five distinct Work Packages (WPs), each targeting specific objectives to deepen our understanding of egg fertilization mechanisms.
In Work Package 1, I combined egg activation/fertilization with advanced microscopy techniques. Central to this effort was the development of high-resolution imaging protocols, essential for the detailed visualization of the egg actin cortex, utilizing both confocal and stereomicroscopy techniques. A key aspect of my methodology was the avoidance of high-salt-containing inactivation media, traditionally used in this field of study. I recognized that such media can potentially slow down the activation process, thereby hindering the observation of its full dynamics. This decision underscored my commitment to capturing the process in its most authentic and undisturbed form.
To counteract the challenge posed by the high mobility of eggs during imaging, I engineered a setup based on egg confinement. This approach allowed for more controlled and precise observations. However, it was imperative to consider the potential mechanosensitive effects of this confinement, particularly its influence on the coupling between the chorion and the plasma membrane of the egg. To address this, and to ensure a comprehensive examination of the process, I also developed a setup for imaging unconfined eggs.
Additionally, a significant part of my work involved developing a framework for the microinjection of dyes and pharmacological drugs into inactivated eggs. This technique was critical for manipulating and observing various intracellular processes during egg activation. To enhance our visualization capabilities, I also undertook the task of creating specialized transgenic zebrafish lines, such as KDEL-mScarlet for imaging structures like the endoplasmic reticulum.
Following the successful integration of Work Package 1, my focus in WP2 was directed towards the comprehensive characterization of the egg actomyosin cortex during the critical phase of egg activation. This phase of the project saw several pivotal achievements, each contributing to our deeper understanding of this process.
One of the primary accomplishments in WP2 was establishing a detailed timeline delineating the changes occurring in the egg cortex surrounding activation. This timeline was instrumental in defining the major growth phases and changes within the perivitelline space and the cortex itself. Such a temporal map provided a clearer understanding of the dynamics at play during the critical moments of egg activation.
Central was the visualization of the interactions between actin, myosin, and calcium within the eggs. This involved not only observing these components in isolation but also understanding their intricate interplay during the activation process. Another key aspect of my approach involved the use of various pharmacological agents, such as 2-APB, Latrunculin-A, and Blebbistatin. These compounds were employed to probe their effects on calcium currents and the actomyosin cortex, allowing me to manipulate and observe the consequent changes.
Furthermore, I delved into the characterization of the Perivitelline fluid (PVF) through advanced proteomics. This not only provided deeper insights into the egg's microenvironment but also will enable us to establish protocols to actively modify PVF properties. Investigating the direct link between the PVF and the cortex is a novel angle that highlights the role of environmental influences on egg activation and development.
The work accomplished in the initial phases has laid a robust foundation for future research in this field. The insights and advancements I have made provide valuable contributions to our understanding of egg activation and fertilization. These findings not only enhance our scientific knowledge but also hold the potential to influence future developments in reproductive medicine.
In Work Package 1, I combined egg activation/fertilization with advanced microscopy techniques. Central to this effort was the development of high-resolution imaging protocols, essential for the detailed visualization of the egg actin cortex, utilizing both confocal and stereomicroscopy techniques. A key aspect of my methodology was the avoidance of high-salt-containing inactivation media, traditionally used in this field of study. I recognized that such media can potentially slow down the activation process, thereby hindering the observation of its full dynamics. This decision underscored my commitment to capturing the process in its most authentic and undisturbed form.
To counteract the challenge posed by the high mobility of eggs during imaging, I engineered a setup based on egg confinement. This approach allowed for more controlled and precise observations. However, it was imperative to consider the potential mechanosensitive effects of this confinement, particularly its influence on the coupling between the chorion and the plasma membrane of the egg. To address this, and to ensure a comprehensive examination of the process, I also developed a setup for imaging unconfined eggs.
Additionally, a significant part of my work involved developing a framework for the microinjection of dyes and pharmacological drugs into inactivated eggs. This technique was critical for manipulating and observing various intracellular processes during egg activation. To enhance our visualization capabilities, I also undertook the task of creating specialized transgenic zebrafish lines, such as KDEL-mScarlet for imaging structures like the endoplasmic reticulum.
Following the successful integration of Work Package 1, my focus in WP2 was directed towards the comprehensive characterization of the egg actomyosin cortex during the critical phase of egg activation. This phase of the project saw several pivotal achievements, each contributing to our deeper understanding of this process.
One of the primary accomplishments in WP2 was establishing a detailed timeline delineating the changes occurring in the egg cortex surrounding activation. This timeline was instrumental in defining the major growth phases and changes within the perivitelline space and the cortex itself. Such a temporal map provided a clearer understanding of the dynamics at play during the critical moments of egg activation.
Central was the visualization of the interactions between actin, myosin, and calcium within the eggs. This involved not only observing these components in isolation but also understanding their intricate interplay during the activation process. Another key aspect of my approach involved the use of various pharmacological agents, such as 2-APB, Latrunculin-A, and Blebbistatin. These compounds were employed to probe their effects on calcium currents and the actomyosin cortex, allowing me to manipulate and observe the consequent changes.
Furthermore, I delved into the characterization of the Perivitelline fluid (PVF) through advanced proteomics. This not only provided deeper insights into the egg's microenvironment but also will enable us to establish protocols to actively modify PVF properties. Investigating the direct link between the PVF and the cortex is a novel angle that highlights the role of environmental influences on egg activation and development.
The work accomplished in the initial phases has laid a robust foundation for future research in this field. The insights and advancements I have made provide valuable contributions to our understanding of egg activation and fertilization. These findings not only enhance our scientific knowledge but also hold the potential to influence future developments in reproductive medicine.
In my 13-month research project, structured into targeted Work Packages, I achieved significant breakthroughs in understanding egg fertilization mechanisms. My efforts focused primarily on the innovative use of zebrafish eggs to study the dynamic role of the egg actomyosin cortex during activation, providing insights beyond the current state of the art in reproductive biology.
My findings have added new dimensions to our understanding of calcium signaling in zebrafish egg activation. I discovered that calcium currents are localized specifically to areas of the egg that are immersed in water. My results showed that even hyperosmotic media initiate egg activation, suggesting that while osmolarity influences cell size changes shortly after activation, it doesn't majorly affect the onset of activation or intracellular signaling. This challenges the prevailing belief about osmotic forces driving egg activation in zebrafish eggs.
A notable observation was the transition of zebrafish eggs from an elastic state to a more pliable condition post-activation. Contrary to initial expectations, this change was not driven by cortex properties but rather by a shift in surface tension due to water immersion. This finding indicates that factors other than traditional biochemical signaling, such as physical properties, play a significant role in egg activation.
Moreover, I observed that destabilizing the actin cortex leads to significant challenges in maintaining cortex integrity during initial PVF growth. This highlights the critical role of the actin cortex in sustaining the structural integrity of the egg, especially during plasma membrane and chorion de-coupling. This relationship could be direct or indirect, possibly through providing stability to the actin cortex itself.
Furthermore, the actin cortex dynamically modulates various stages of egg activation, including potential exocytotic and endocytotic processes. A key discovery was that actin dots form after the primary phase of PVF expansion, indicating a significant role for the actin cortex in the later stages of egg activation. This suggests a complex involvement of the actin cortex beyond its initial functions, extending to the egg's response to mechanical stresses and structural remodeling following activation.
To maximize the impact and advance the uptake of these findings, continuous research is essential, particularly to delve deeper into the mechanistic details that this study has brought to light. I have already initiated further investigations into the potential mechanosensitivity of the egg and the expansion of the perivitelline fluid (PVF) as a form of pressure buildup, which could be pivotal in determining the course of embryonic development. This exploration into uncharted territories of reproductive biology could set the stage for further developments in reproductive health.
My findings have added new dimensions to our understanding of calcium signaling in zebrafish egg activation. I discovered that calcium currents are localized specifically to areas of the egg that are immersed in water. My results showed that even hyperosmotic media initiate egg activation, suggesting that while osmolarity influences cell size changes shortly after activation, it doesn't majorly affect the onset of activation or intracellular signaling. This challenges the prevailing belief about osmotic forces driving egg activation in zebrafish eggs.
A notable observation was the transition of zebrafish eggs from an elastic state to a more pliable condition post-activation. Contrary to initial expectations, this change was not driven by cortex properties but rather by a shift in surface tension due to water immersion. This finding indicates that factors other than traditional biochemical signaling, such as physical properties, play a significant role in egg activation.
Moreover, I observed that destabilizing the actin cortex leads to significant challenges in maintaining cortex integrity during initial PVF growth. This highlights the critical role of the actin cortex in sustaining the structural integrity of the egg, especially during plasma membrane and chorion de-coupling. This relationship could be direct or indirect, possibly through providing stability to the actin cortex itself.
Furthermore, the actin cortex dynamically modulates various stages of egg activation, including potential exocytotic and endocytotic processes. A key discovery was that actin dots form after the primary phase of PVF expansion, indicating a significant role for the actin cortex in the later stages of egg activation. This suggests a complex involvement of the actin cortex beyond its initial functions, extending to the egg's response to mechanical stresses and structural remodeling following activation.
To maximize the impact and advance the uptake of these findings, continuous research is essential, particularly to delve deeper into the mechanistic details that this study has brought to light. I have already initiated further investigations into the potential mechanosensitivity of the egg and the expansion of the perivitelline fluid (PVF) as a form of pressure buildup, which could be pivotal in determining the course of embryonic development. This exploration into uncharted territories of reproductive biology could set the stage for further developments in reproductive health.