The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes

Abstract

Porcine animal models are used to advance our understanding of human physiology. Current research is also directed at methods to produce transgenic pigs. Cryobanking gametes and embryos can facilitate the preservation of valuable genotypes, yet cryopreserving oocytes from pigs has proven very challenging. The current study was designed to understand the effects of anisotonic solutions on in vitro matured porcine oocytes as a first step toward designing improved cryopreservation procedures. We hypothesized that the proportion of oocytes demonstrating a normal spindle apparatus and in vitro developmental potential would be proportional to the solution osmolality. Oocytes were incubated for 10 min at 38 degrees C in various hypo- or hypertonic solutions, and an isotonic control solution and then assessed for these two parameters. Our results support the hypothesis, with an increasing proportion of spindles showing a disrupted structure as the levels of anisotonic exposure diverge from isotonic. Only about half of the oocytes maintained developmental potential after exposure to anisotonic solutions compared to untreated controls. Oocyte volume displayed a linear response to anisotonic solutions as expected, with an estimated relative osmotically inactive cell volume of 0.178. The results from this study provide initial biophysical data to characterize porcine oocytes. The results from future experiments designed to determine the membrane permeability to various cryoprotectants will allow predictive modeling of optimal cryopreservation parameters and provide a basis for designing improved cryopreservation procedures.

Figure 1

The steps in the procedure used in image analysis are shown from left to right. The first panel shows an original photograph. A threshold process, with the oolemma intensity chosen as the threshold level, was applied to the original image, isolating pixels based upon their grayscale value. The result is shown in the second panel. The next process involved filling in areas that were completely surrounded by black pixels (panel 3). Finally, all objects smaller than a minimum size (chosen as 4000 pixels) were rejected from the image, leaving only the area originally occupied by the oocyte (panel 4). The radius of a circle containing an equivalent area was computed by the software, as shown. The volume of the oocyte was calculated using this value, assuming spherical geometry.

Figure 2

The relationship between the cell volume of in vitro matured porcine oocytes and the solution osmolality (Miso/M) is shown. Data points represent the mean ± SEM.

Figure 3

Various morphologies of MII spindles are shown. Normal spindle morphology is represented by the images in panels A and B, with near spherical shaped spindles and chromosomes aligned along the metaphase plate. Panel C shows a spindle which has chromosomes dislocated from the metaphase plate, with some located near one of the spindle poles. Panel D shows a spindle after oocyte activation, with the chromosomes in a late-anaphase configuration, being located at opposite sides of the spindle. The images in Panels E and F show abnormal spindle configurations. The spindle in Panel E was highly disrupted, with reduced microtubule density as well as displaced chromatin. The spindle in Panel F shows the development of multiple spindle poles after treatment, with chromosomes being displaced toward these multiple poles.

Figure 4

The proportions of oocytes having a normal spindle configuration (with exact 95% confidence intervals; left panel) and in vitro developmental potential (± SEM; right panel) after equilibration with solutions of various osmolalities are shown. The respective cell volumes have been added as a second x-axis for comparison. The cell volume response does not follow the expected Boyle van’t Hoff relationship as expected in mammalian oocytes at hypotonic osmolalities, as the cell is inhibited from swelling due to the presence of the zona pellucida. Thus the volume data for the hypotonic treatments beyond 225 mOsm were not included on this scale. We have plotted the X-axis as a logarithmic scale to make the data easier to visualize, and not due to any assumption about logarithmic relationship between the variables.