(F-208) Apparent Young’s Modulus of Xenopus Laevis Oocytes Measured Using Atomic Force Microscopy at Different Set Points

Introduction:: Mechanical properties, such as apparent Young’s modulus (YM), of live cells have been extensively studied. The values reported in the literature span three orders of magnitude ranging from 0.1 kPa to 100 kPa. This variation could be attributed to the use of different experimental techniques, instrument settings, and cell types. For example, oocytes possess a thick vitelline membrane (VM) that covers the plasma membrane (PM), protects the cell, and gives shape to the oocyte. In addition to its structural role, VM also creates a barrier against sperm penetration during fertilization. In this study, we compared apparent YM of the oocytes with an intact VM measured at the animal and vegetal poles using three different setpoints. In addition, several measurements were performed on oocytes where the VM had been removed.

Materials and Methods:: Live unfertilized Xenopus Laevis oocytes from a commercial vendor (Ecocyte) were used for all measurements. AFM force spectroscopy was performed on the oocytes with and without VM in this study. The apparent YM were measured on the animal pole of 26 oocytes and the vegetal pole of 20 oocytes, both with intact VM. Additionally, YM measurements were taken on 4 oocytes without the VM. For the experiments without the VM, the oocytes were soaked in Vitelline Removing Solution (Ecocyte) followed by manual removal of the VM with sharp forceps. A Bruker JPK NanoWizard 4 BioAFM system (AFM) was used for imaging and force spectroscopy measurements. For elastic property measurements cantilever with cylindrical tip, 10 μm end radius and nominal spring constant of 0.25 N/m was used (SAA-SPH-10UM, Bruker). Ultra-sharp probe with 1 nm end tip radius and nominal spring constant of 0.35 N/m (PEAKFORCE-HIRS-FA, Bruker) was used for imaging. The force-displacement (FD) curves were collected on 50x50 μm areas with 3x3 grid at 2 μm/s Z speed, 5 μm Z length and three set point values of 3, 5, and 10 nN. AFM imaging was performed on a region that measured 50x50 μm with the following settings: setpoint of 0.15 nN, Z Length of 3000 nm, Z speed of 75 μm/s, and resolution 128x128 pixels. Oocytes were immobilized during the measurements using a custom sample holder laser cut from 0.75-mm thick PETG sheet.


Results, Conclusions, and Discussions:: Figure 1 shows the AFM image of a live oocyte surface obtained in the QI mode. An interconnected network of protein fibers with thicknesses up to 1 μm is visible. FD curves were acquired from the oocytes with and without the VM. The Hertz model was used to process the results. Apparent average YM of oocytes with VM was found to be in the range of 0.7 to 4 kPa (1.66 ± 0.65 kPa), and in the case of oocytes without the VM - less than 0.1 kPa (0.06 ± 0.03 kPa). Analysis of variance (ANOVA) was performed on average values of YM obtained for each oocyte. The results show that the difference between the YM obtained using 3, 5, and 10 nN set points are marginally statistically significant in the vegetal pole (p = 0.0363) and statistically insignificant in the animal pole (p = 0.0921). The apparent YM measured on the vegetal pole is stiffer than the animal pole and the difference is statistically significant (p = 0.000019 for 3 nN, p = 0.0011 for 5 nN, p = 0.0024 for 10 nN).

In this study, AFM was used to measure apparent YM of Xenopus oocytes. Performing measurements on oocytes without the VM is challenging because after removal of the VM they lose their structural integrity rapidly. Preliminary results show that the oocyte surface becomes significantly softer after removing the VM. Our observations point to VM being the primary structural component maintaining cell shape.


Acknowledgements (Optional): : This material is based upon work supported by the National Science Foundation under grants No. CMMI-2011220 and No. DBI 2117045.

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