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        Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes
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        Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes
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        Ultrastructural Imaging Analysis of the Zona Pellucida Surface in Bovine Oocytes
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The aims of the present study were to: (i) evaluate the ultrastructural differences in the zona pellucida (ZP) surface between immature and mature bovine oocytes, and (ii) describe a new objective technique to measure the pores in the outer ZP. Intact cumulus–oocyte complexes (COCs) obtained from a local abattoir were immediately fixed (immature group) or submitted to in vitro maturation (IVM) at 38.5 °C for 24 h in a humidified atmosphere of 5% CO2 in air (mature group). Oocytes from both groups were morphologically evaluated via Scanning Electron Microscopy (SEM) and the images were processed in the Fiji/ImageJ software using a new objective methodology through the Trainable Weka Segmentation plugin. The average number of pores in ZP was greater (p < 0.05) in the mature group than the immature group. However, the size and circularity of pores in ZP did not differ (p > 0.05) between groups. In conclusion, it has been shown that the number of pores highlighted the main ultrastructural change in the morphology of the ZP surface of bovine oocytes during the IVM process. We have described an objective method that can be used to evaluate ultrastructural modifications of the ZP surface during oocyte maturation and early embryo development.


The mammalian zona pellucida (ZP) is an extracellular coat that surrounds growing and matured oocytes, and early embryos (Dunbar et al., 1994; Epifano & Dean, 1994). The ZP has several functions such as binding spermatozoa in a species-specific manner, blocking polyspermy, preventing the dispersion of blastomeres during preimplantation development, and protecting the embryo during the early stages of development (Wassarman, 1990; Epifano & Dean, 1994). However, the ZP has a dynamic structure that may change according to species, origin, embryonic stage, and other intrinsic and extrinsic factors (Zhao et al., 2014). Previous studies have shown that the ZP of several species (cat, rabbit, hamster, mouse, rat, opossum, and cow) has a complicated and porous network structure (Dudkiewicz & Williams, 1977). The porous structure might be the result of foot-like cytoplasmic branches from granulosa cells of the surrounding corona radiata, penetrating the ZP to come in close contact to the plasma membrane of the oocyte during oogenesis (Magerkurth et al., 1999). When the porous surface is compared among species, the largest pores are observed in the ZP of the rabbit and the cat, and the smallest in the cow; the pores are large at the outer surface of the ZP and decreasing in size centripetally (Dudkiewicz & Williams, 1977). Moreover, the spongy and lattice-like appearance discovered in the ZP of bovine oocytes is associated with the maturation process, the expansion of cumulus cells, and the maturation of the ZP after fertilization (Suzuki et al., 1994). In addition, the morphological appearance (Held et al., 2012), number, and diameter of ZP pores are predictive of the quality of the oocytes and subsequent embryonic development (Santos et al., 2008; Choi et al., 2013). Differences have been observed in respect to the average diameter of ZP pores (Vanroose et al., 2000; Santos et al., 2008; Choi et al., 2013), which may be due to the source of oocytes (slaughterhouse versus ovum pick-up), conditions of in vitro maturation (IVM), methods of analyses, or a combination of these factors.

The studies neither have an unequivocal standard of definition of the ZP structure nor use the same method of image analysis. In fact, a reliable method to evaluate whether variations of ZP ultrastructure are related or not to oocyte maturation stage or the various phenotypes observed may correspond to artifacts (e.g., strong solutions of ethanol or glutaraldehyde may cause shrinkage of ZP gel structure generating relevant artifacts) still not described. Although oocytes may be treated differently within the experiments, particular care should be given when preparing the samples for scanning electron microscopy (SEM) and standardized examination should be used to correctly interpret and compare the morphology of the ZP as this will certainly reduce or even avoid ultrastructural artifacts (Familiari et al., 2006). However, an objective description of the ultrastructural characteristics of ZP, comparing immature and mature stages of bovine oocytes performed by a simple computational analysis of the digital images, is still valuable. Therefore, the aims of the present study were to: (i) evaluate the ultrastructural differences in the ZP surface between immature and mature bovine oocytes and (ii) describe a new technique to measure the ZP pores.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.

Collection of Ovaries and Oocytes

Bovine ovaries (n = 38) were collected from a local abattoir after slaughter and transported to the laboratory within 2 h in a thermal bottle containing a physiological saline solution (0.9% NaCl) at approximately 35 °C. In the laboratory, ovaries were washed three times with a saline solution containing 50 µg/mL gentamicin, and cumulus–oocyte complexes (COCs) were retrieved from follicles 2–8 mm by aspiration using an 18-gauge needle attached to a 10 mL syringe. COCs that were surrounded by compact multiple layers of cumulus cells were used for the study and were washed three times in TL-HEPES medium [114 mM sodium chloride, 3.2 mM potassium chloride, 2 mM sodium bicarbonate, 0.34 mM sodium biphosphate, 10 mM sodium lactate, 0.5 mM magnesium chloride, 2.0 mM calcium chloride, 10 mM HEPES, 4 mg/mL BSA fraction V, and 50 µg/mL gentamicin]. After washing, COCs were randomly submitted to fixation (immature group) or IVM (mature group) processes.

Experimental Design

This study was designed to determine the effect of IVM on the ultrastructural characteristics in the outer surface of the ZP of bovine oocytes. After retrieval, immature COCs (n = 40, immature group) were immediately fixed and processed for SEM, whereas 110 COCs were submitted to IVM. After IVM, matured oocytes (n = 40, mature group) were processed for SEM and the remaining oocytes (n = 70) were fixed and stained to assess meiotic progression. Four replicates were performed.

In Vitro Maturation

COCs (n = 110) were subjected to IVM in four-well dishes (Thermo Fisher Scientific, Waltham, MA, USA) containing 500 µL maturation medium [TCM-199, supplemented with 10% (v/v) fetal calf serum (Gibco BRL, Life Technologies, Grand Island, NY, USA), 1 µg/mL estradiol-17ß, 1 µg/mL follicle-stimulating hormone (Folltropin-V®, Bioniche Animal Health, Belleville, Ontario, Canada), 0.5 mM cysteamine, and 0.2 mM sodium pyruvate] for 24 h at 38.5 °C and 5% CO2 in humidified air. After IVM, expansion of the cumulus cells was evaluated in all oocytes under a stereomicroscope before denudation.

Nuclear Assessment

Only COCs from IVM were submitted for nuclear assessment to determine the maturation rate according to the meiotic stage. After IVM, oocytes were completely denuded using a mechanical process to remove cumulus cells by repeated pipetting in TL-HEPES medium (without BSA), and dispersion of cumulus was verified using a stereoscopic microscope. Oocytes were fixed in ethanol: acetic acid (3:1 v/v) for at least 48 h. Then, fixed oocytes were stained with aceto-orcein stain (1% orcein in 45% acetic acid) and evaluated under an optical microscope (×400 original magnification; Nikon, Tokyo, Japan). The oocytes were evaluated for the stage of nuclear maturation as: germinal vesical, metaphase I, or metaphase II (Hewitt et al., 1998).

Scanning Electron Microscopy and Image Processing

Oocytes were denuded as above mentioned, and prepared for SEM as previously described (Moreira da Silva & Metelo, 2005) with minor modifications. Briefly, oocytes were placed in fixation medium (2.5% glutaraldehyde [v/v] and 0.1 mol/L sodium cacodylate buffer) for 1 h at 4 °C. Then, washed with 0.1 mol/L sodium cacodylate buffer and kept in the buffer for 1 h at 4 °C, followed by washing in distilled water for 5 min. Thereafter, oocytes were dehydrated with increasing concentrations of ethanol at room temperature. After dehydration, the oocytes were CO2 critical point dried in a stainless chamber (Denton Vacuum DCP-1, Denton Vacuum, LLC, Moorestown, NJ, USA), mounted onto aluminum stubs, and coated with 20 nm of gold by sputtering (Denton Vacuum Desk II,  Denton Vacuum, LLC, Moorestown, NJ, USA). After sample processing, 24 oocytes (12 per group) were selected for the SEM analysis under the following criteria: the presence of no cumulus cells and any artifacts surrounding the oocytes that could jeopardize the quality of pictures and measurements of the ZP pore.

Photomicrographs of the ZP of oocytes were taken to access and count the number of pores in a selected region of interest (12 × 8 µm) within the image area (20 × 15 µm) in a JEOL JCM-6000 PLUS SEM (JEOL USA, Inc., Peabody, MA, USA) using on-scope magnifications from ×700 to ×7000. Clear images of bovine oocytes were saved to disk for further analysis of the ultrastructural surface of the ZP. Measurements on each SEM image of the ZP (number, area, and circularity of pores) were determined using the public domain Fiji/ImageJ image processing software (Schindelin et al., 2012). The region of interest in the image (Fig. 1a), corresponding to pores in the ZP, was selected by the Trainable Weka Segmentation plugin (; Arganda-Carreras et al., 2017). The pores were components of the image with values ranging from 0 to 50 pixels with morphological characteristics delimited to holes with nearly smooth and compact aspect (Vanroose et al., 2000; Familiari et al., 2006). The area occupied by a pore was selected and classified as a group (class 1); while the remaining components in the image are selected and classified within another group (class 2). Classification parameters are stored in a file and used to segment all images maintaining an identical classification model for each case. The output is a mask (Fig. 1b) containing only the pores present in the ZP of the original image (Fig. 1c, outcome of a and b superimposed). To determine the measurements (number, area, and circularity) of the pores, the “Analyze Particles” software tool was used choosing the parameters of identification, size (≥0.01 µm2; Vanroose et al., 2000) and circularity (between 0.4 and 1; Fig. 1d). Lastly, mask and plane were merged to better image the pores analyzed (Fig. 1e).

Fig. 1. Representative digital images illustrating the selection process of the external surface of the ZP pores in IVM bovine oocytes in the Fiji/ImageJ image processing software. a: SEM micrographs and image analysis of SEM; (b) first-mask, segmentation output locating pores; (c) second-mask selecting pores in ZP (a and b superimposed), (d) plane illustrating only the selected pores in ZP; and (e) final mask with c and d images superimposed. The scale bar represents 2 µm on magnification of ×7,000.

Statistical Analysis

Data were tested for normal distribution and homogeneity of variance using the Kolmogorov–Smirnov and Levene's test before statistical procedures. Comparisons between groups for all measurements of the ZP pores were performed by one-way ANOVA. All the statistical analyses were done using SAS statistical software package (SAS Inst. Inc.; Cary, NC, USA). Data were expressed as mean ± SEM, unless otherwise indicated. A probability of p < 0.05 indicated that a difference was significant.


The maturation rate of oocytes submitted to IVM was 89.0 ± 2.0%. With respect to the ultrastructural characteristics, all immature oocytes showed a ZP surface that was characterized by a rough, irregular fibrous network with an uneven distribution and tight pores, crevices, and depositions of biological material (Figs. 2a, 2c). Mature oocytes displayed a porous zona structure with typical fine-meshed reticular pores; the pore shape was circular or elliptical and arbitrarily distributed (Figs. 2b, 2d). The average number of pores in the ZP was greater (p < 0.05) in the mature oocytes than in the immature oocytes (Table 1). However, area, diameter, and circularity did not differ (p > 0.05) between groups.

Fig. 2. Scanning electron micrographs of the external surface of the ZP of (a) immature and (b) IVM bovine oocytes. The ultrastructural characteristics of ZP pores of (c) immature and (d) IVM oocytes can be observed in higher magnification. The bars represent 20 µm and magnification of ×750 and ×1,300 (a and b figures, respectively), and 2 µm with magnification ×7,000 (c and d).

Table 1. Ultrastructural characteristics of the pores in the ZP of immature and IVM bovine oocytes analyzed by Trainable Weka Segmentation plugin in Fiji/ImageJ software.

* Circularity was calculated in Fiji/ImageJ using the following formula: circularity = 4Pi(area/perimeter^2); a circularity value of 1.0 indicates a perfect circle. As the value approaches 0.0, it indicates an increasingly elongated polygon. Different letters in the rows indicate statistical significance (p < 0.05).


To our knowledge, this is the first study to directly compare the ultrastructure of the outer ZP pores surface of immature and in vitro matured bovine oocytes. The present study has shown that morphological changes occur in the outer ZP surface of immature bovine oocytes after the IVM process. The ZP pores of immature bovine oocytes have shown a very irregular shape, with uneven distribution of numerous pores, crevices, and projections. After IVM, although the thin structure network becomes thinner and the holes seem to be shallower, the number of pores increased. Our findings are corroborated with previous studies (Riddell et al., 1993; Suzuki et al., 1994) demonstrating that the outer surface of the ZP of immature bovine oocytes has a thin structure characterized by a wide network and deep pore holes (Suzuki et al., 1994). Previous studies using SEM analysis reported that the average number of ZP pores and the mean diameter (in nm) of the outer pore of bovine oocytes matured in vitro were (pores/diameter): 42/428 (Choi et al., 2013), 55/500 (Santos et al., 2008), and 1511/182 (Vanroose et al., 2000), determined in an area of interest of 96, 40.96, and 5,000 µm2, respectively. The smaller diameter and greater number of ZP pores are related to superior oocyte quality as well as to a higher rate of blastocyst formation (Santos et al., 2008; Choi et al., 2013). However, fertilized oocytes did not present meshes (network of pores) on the ZP, probably due to the fusion of several layers of the network during the fertilization process (Vanroose et al., 2000). Therefore, the variations observed in the characteristics of the ZP surface may be due to its 3D network of crossing filaments and the spongy or compact appearance (Familiari et al., 2006) and the different oocyte maturation stages among studies.

The present study has developed a new methodology combining the SEM and Fiji/ImageJ software analysis for objectively evaluating the ultrastructure of the outer ZP surface. The method allowed us to select the pores according to pre-established criteria (e.g., number, area, and circularity), avoiding misinterpretation. Therefore, the number of pores in the outer ZP surface was the only variation we observed between immature and in vitro matured bovine oocytes. Ultrastructural characteristics of the ZP have been related to the developmental competence of bovine oocytes and fertility potential (Santos et al., 2008). Other factors may change the ultrastructure characteristics of the ZP of oocytes such as coculture with denuded oocytes, retrieval by in vivo ovum pick-up versus abattoir ovaries, and culture medium (Choi et al., 2013). Therefore, given the importance of measurements regarding the number and diameter of pores in ZP and the relationship with oocyte quality and fertility, the present method may facilitate future studies to understand the effect of external factors on oocyte development using an objective technique.


In the present study, we described a new objective methodology to analyze the ultrastructure of the outer ZP surface and demonstrated the ultrastructural differences in the ZP surface between immature and mature bovine oocytes. The IVM process causes changes in the ultrastructure of the outer ZP and an increase in the ZP pore number when compared to immature oocytes. Therefore, differences observed in the number of ZP pores during oocyte maturation process highlight the importance of the starting point for evaluations to assess fertilization and early embryonic development.

Author ORCIDs

Gustavo D.A. Gastal, 0000-0002-5317-2207


The authors are grateful to the slaughterhouse Tacuarembó of Marfrig group S.A. for providing the specimens and the Microscopy Unity in the Faculty of Sciences of the University of the Republic, Uruguay (UDELAR) for assistance.


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