Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality

Theriogenology 80 (2013) 498–506

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Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality Matthew O. Lunn, Shirley J. Wright* Department of Biology, University of Dayton, Dayton, Ohio, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 March 2012 Received in revised form 15 May 2013 Accepted 19 May 2013

The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from lowquality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canineassisted reproduction. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Oocyte Zona pellucida Scanning electron microscopy Canine Dog

1. Introduction During fertilization, the sperm binds and penetrates the zona pellucida (ZP), an elastic extracellular matrix surrounding the mammalian oocyte and preimplantation embryo. Zona function is critical not only for oocyte viability and growth but also for several essential steps of fertilization, including triggering the acrosome reaction, speciesspecific sperm binding, and prevention of polyspermy [1,2]. In addition, the zona protects the preimplantation * Corresponding author. Tel.: þ937 229 2857; fax: þ937 229 2021. E-mail address: [email protected] (S.J. Wright). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.05.013

embryo during its passage in the oviduct to the uterus and promotes embryo survival [1,2]. The zona matrix is composed of three or four glycoproteins referred to as ZP1, ZPA/ZP2, ZPC/ZP3, and ZPB/ZP4 [3]. All mammalian zonae examined to date have ZPA/ZP2 and ZPC/ZP3, but vary in ZP1 and ZPB/ZP4 content [3]. For example, both ZP1 and ZPB/ZP4 are present in the zonae of rats, hamsters, horses, macaques, chimpanzees, and humans [3–5]. In contrast, zonae of domestic animals (dog, cat, rabbit, pig, and cow) lack ZP1, but contain ZPB/ZP4 protein [3]. Unlike any other mammal, the mouse zona contains ZP1, but not ZPB/ZP4 [3,6]. Mouse oocytes synthesize and secrete ZP proteins, which form an insoluble

M.O. Lunn, S.J. Wright / Theriogenology 80 (2013) 498–506

extracellular matrix around the oocyte [6]. Mouse ZPA/ZP2 and ZPC/ZP3 are sufficient to form a functional zona matrix, with their cytoplasmic tails preventing premature oligomerization in the ooplasm [7]. In dogs, this problem was solved by having the oocyte synthesize ZPA/ZP2 and the granulosa cells produce ZPC/ZP3 and ZPB/ZP4 [8]. Lack of ZP2 or ZP3 results in infertility in mice [9]. Certain cases of human infertility are linked to problems with the ZP, including crosslinking of zona glycoproteins, causing zona hardening [1]. Therefore, it is important to understand ZP structure. Zona proteins assemble to form a complex, threedimensional fibrous network with many pores [10,11]. It has been proposed that the mesh-like arrangement of ZP fibers has biological importance in assisting sperm to become oriented appropriately for fertilization [12–14]. In addition, species differences in ZP protein composition have led to differences in ZP structure and potentially the mechanism of sperm interaction with the ZP [5]. Studying zonae of domestic animals could provide new insight into its function and sperm interaction with the ZP [5]. When viewed with scanning electron microscopy (SEM), the ZP surface appears spongy in most mammals. However, when comparing species, ZP morphology varies with respect to several parameters, including ZP thickness, pore characteristics (density, size, shape, and depth of pores), and thickness of the fibers surrounding the pores [10,15]. In addition, a heterogeneous morphology of the external ZP surface has been reported for mouse, cow, pig, goat, and human oocytes [13,14,16–19]. Structural heterogeneity of the ZP has been linked to meiotic maturation in oocytes of several mammals, including humans, although this is controversial in the literature. For example, variation in ZP structure has been linked to oocyte maturation in the mouse, dog, goat, and human [12,14,16,17,20]; however, other researchers using mouse, hamster, pig, or human oocytes did not report an association [21–23]. Moreover, ZP structure of oocytes matured in vitro differed from that of in vivo–matured oocytes in several species [14,16,18,24]. However, Suzuki et al. [25] reported that the porcine ZP surface was unaltered during in vitro maturation. Whether species variations or differences in specimen preparation account for this is unclear. Canine reproductive biology is distinct from most mammals. In the adult dog, each ovarian oocyte is maintained at meiotic prophase I (germinal vesicle (GV) stage) and is surrounded by the ZP and cumulus cells [26–28]. During follicle growth, the ooplasm matures acquiring dark lipid droplets, mitochondrial clouds, and large parallel or concentric strands of smooth endoplasmic reticulum in great abundance compared with oocytes of other mammals [26,29]. On hormonal cues, a recruited subset of primary oocytes grows and the largest become meiotically competent [26–30]. They are ovulated at the GV stage as cumulusoocyte complexes (COCs) [26–31]. The oocytes resume meiosis within the lumen of the oviduct and are fertilized at metaphase II [26–31]. It is unclear whether in preparation for ovulation, the canine ZP undergoes a maturational process in parallel with ooplasmic maturation. Therefore, the objective of the present study was to examine ZP surface morphology of large ovarian canine oocytes with

499

SEM to determine whether they exhibit similar variations in the ZP surface as reported for other mammals, and if ZP morphology corresponds to oocyte viability, COC quality and age of the canine oocyte donor. 2. Materials and methods 2.1. Ovary collection Ovaries in the ovarian bursa were obtained from 56 healthy, domestic dogs of various breeds (including mixed breeds) at random stages of the estrous cycle (including anestrous) during routine ovariohysterectomy at a local veterinary practice. The dogs averaged 2.1  0.3 years with the youngest at 3 months (n ¼ 1) and the oldest at 9 years (n ¼ 1). Ovaries were immediately placed in PBS at 4  C. 2.2. Collection of COCs Each ovary was dissected from the ovarian bursa and rinsed in PBS before repeated slicing to release COCs, as described [32]. Briefly, COCs were placed in fresh PBS and graded using a stereomicroscope according to Hewitt and England [33]. Unless stated otherwise, high-quality (grade 1) COCs, which had two or more layers of cumulus cells that completely surrounded a darkly pigmented, homogeneous ooplasm were used for the study. 2.3. Oocyte collection To release oocytes from COCs and reveal the ZP surface, cumulus cells were mechanically removed by repeated pipetting with the Stripper using a 150-mm Stripper tip (MidAtlantic Diagnostics, Inc., Marlton, NJ, USA) [32]. Cumulus-free, zona-intact oocytes were placed in fresh PBS before use. Oocytes were collected from the largest COCs only, because smaller COCs were resistant to mechanical stripping with our protocol. A total of 579 oocytes were used for the study. 2.4. Oocyte diameter Because it has been reported that age of the dog oocyte donor may affect oocyte quality [34], we ascertained whether oocyte donor age influenced oocyte diameter, which relates to acquisition of meiotic competence [30]. To that end, 402 oocytes were collected from high-quality COCs obtained from 44 dogs with an average age of 2.2  0.3 years. All oocytes collected on any given day were prepared for SEM together (as described in Section 2.5). Each oocyte was grouped according to reproductive age of the dog ovary donors: “prepubertal” (<6 months, n ¼ 1 donor), “juvenile” (6–11 months, n ¼ 14 donors), “mature” (12–48 months, n ¼ 22 donors), and “old” (>48 months, n ¼ 7 donors), and there were 3, 155, 184, and 60 oocytes in these age groups, respectively. The diameter of each oocyte (including the ZP) was measured from scanning electron micrographs by averaging two measurements made perpendicular to each other for each oocyte. In rare instances, a diagonal axis was taken if one of the two axes was obscured by cumulus cells.

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2.5. Scanning electron microscopy Oocytes collected each day (usually from one dog donor) were prepared for SEM, according to Lunn and Wright [32]. Briefly, oocytes were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 at 4  C for 1 hour, washed, dehydrated in a graded series of ethanol, critical point dried, mounted, and sputter coated with gold. Oocytes were viewed under high vacuum with a JEOL-5800LV scanning electron microscope (JEOL USA Inc., Peabody, MA, USA) at low (1000) and high (4000) magnification. If an oocyte became cropped because it was too large, a lower magnification (900 or 950) image was taken. To avoid distortion due to curvature of the oocyte, care was taken at high magnification so that each image was restricted to the top central region of the oocyte. Digital images were stored on a computer hard drive and printed as 5  7" electron micrographs. Architecture of the ZP surface was assessed for each oocyte by first observing the low magnification electron micrograph for overall ZP appearance (smooth, rough) and the corresponding high magnification electron micrograph for specific features of ZP surface morphology (pores, fibers). Pore sizes in the ZP were determined from the 4000 scanning electron micrographs by measuring the largest diameter of the pores.

processed as a group. To assess plasma membrane integrity and thus oocyte viability, oocytes were stained with 10 mM PI in PBS for 10 minutes at 37  C in the dark [36,37]. Stained oocytes were viewed with a 4 NA 0.13 objective lens and a HC Red filter set using an Olympus BX51 fluorescence microscope fitted with an Olympus DP71 digital camera. Oocytes with disrupted plasma membrane integrity fluoresced red when exposed to PI and were scored as PIpositive (dead), whereas viable oocytes did not fluoresce and were scored as PI-negative (live) [36,37]. The two groups of oocytes from each canine donor were then prepared simultaneously for SEM and oocyte diameters were determined as described in Sections 2.4 and 2.5. 2.6.3. Experiment 3: effect of COC quality on ZP structure It has been shown that the quality of COCs affects meiotic competence [28]. To determine if COC quality also affects ZP morphology, 73 COCs were isolated from four dogs aged 1.2  0.3 years and sorted into two groups: highquality COCs (grade 1, n ¼ 37 oocytes), as described in Section 2.2, and low-quality COCs (grade 2), which had incomplete layers of cumulus cells and heterogeneous or lightly pigmented ooplasm [33]. After isolation from COCs, both oocyte groups from each dog were processed simultaneously for SEM, and oocyte diameters were determined as described in Sections 2.4 and 2.5.

2.6. Experimental design 2.7. Statistical analysis Three experiments were conducted to analyze the structure of the canine ZP. Oocytes collected from each dog were considered a replicate. Experiment 1 had 44 replicates (44 dogs) and 402 oocytes. Experiment 2 had eight replicates (eight dogs) and 104 oocytes divided into two groups: live (n ¼ 47) and dead (n ¼ 57) oocytes (see Section 2.6.2). Experiment 3 had four replicates (four dogs) and 69 oocytes divided into two groups: high-quality oocytes (n ¼ 35) and low-quality oocytes (n ¼ 34; see Section 2.6.3). 2.6.1. Experiment 1: morphology of the canine ZP surface The ZP external surface of 402 high-quality oocytes collected, as briefed in Section 2.4, was viewed with SEM as described in Section 2.5 to determine whether the canine zona matrix has a heterogeneous surface morphology. In addition, because age of the oocyte donor affected ZP surface characteristics in mice [35], ZP morphology was analyzed in oocytes grouped by age of the dog oocyte donor, using the age grouping described in Section 2.4. 2.6.2. Experiment 2: effect of oocyte viability on ZP structure Structural changes of the ZP have been reported for degenerating oocytes [11,17,35]. To determine whether nonviable oocytes have a different ZP than viable oocytes at the time of processing and whether our protocol affected oocyte viability and ZP morphology from the time of collection, ultrastructure of the outer ZP surface was examined with regards to oocyte viability that was detected with propidium iodide (PI) staining and conventional fluorescence microscopy. Zona-intact oocytes (n ¼ 104) from high-quality COCs harvested from eight dogs with an average age of 2.0  0.3 years were used for Experiment 2. Oocytes collected from each dog were simultaneously

Data were expressed as mean values  standard error of the mean. To compare oocyte diameters with canine age or ZP type, a one-way ANOVA with a Tukey’s multiple comparison post-test was used. A Kruskal-Wallis test with a Duncan’s multiple comparison post-test were used to analyze nonparametric data. A two-way ANOVA with a Bonferroni multiple comparison post-test were used to compare oocyte diame-ters with oocyte characteristics: viability (living vs. dead) and COC grade (high- vs. lowquality). The chi-squared test was used to compare ZP types with oocyte characteristics, whereas Fisher’s exact test was used to compare grouped ZP types (I/II and III/IV) with reproductive age groups and oocyte characteristics. Data were considered significant when P < 0.05. Statistical evaluation was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). 3. Results 3.1. Experiment 1: morphology of the canine ZP surface The diameter of high-quality, zona-intact canine oocytes (n ¼ 402) used to assess the morphology of the canine ZP surface was measured from scanning electron micrographs and averaged 86.7  0.6 mm, in agreement with previous studies of high-quality canine oocytes [29,38,39]. Oocytes from old dogs had the largest diameter (95.2  1.3 mm). Oocytes from mature dogs had a significantly larger diameter (88.5  0.7 mm) than oocytes from juvenile dogs (81.5  0.8 mm). It was difficult to collect cumulus-free oocytes from the prepubertal stage, and our procedures yielded three oocytes from only one prepubertal dog. The average

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diameter (73.9  12.7 mm, mean  SD) of these oocytes was smaller than oocytes from only the old dogs (P < 0.05). The architecture of the outer ZP surface of 402 oocytes from 44 dogs was investigated using SEM. By imaging oocytes at low (1000) and high (4000) magnification, four surface morphologies of the canine ZP were distinguished and categorized as ZP types I, II, III, and IV (Fig. 1). The ZP type I was very smooth with a “melted” appearance at low magnification (Fig. 1A) and was amorphous with very few small pores that were visible at only high

501

magnification (Fig. 1A0 ). These pores were usually <0.5 mm in diameter and randomly spread out across the ZP at low density. The ZP type II was relatively smooth at low magnification (Fig. 1B), but at high magnification it appeared fenestrated due to many round or elliptical pores at high density (Fig. 1B0 ). The pores were often conical and/ or bifurcated as they neared the oolemma. In addition, ZP type II was multilayered (Fig. 1B0 ). The ZP type III had a rough or uneven surface at low magnification (Fig. 1C). At high magnification, ZP type III was a rough and spongy

Fig. 1. Variation in the morphology of the canine ZP. Scanning electron micrographs of the external surface of the ZP of high-quality canine oocytes at low (A–D) and high (A0 –D0 ) magnification showing the four ZP types. ZP type I (A, A0 ), type II (B, B0 ), type III (C, C0 ), and type IV (D, D0 ). Cumulus cells are visible to the right of the oocyte in A. Scale bars: 20 mm in A–D, 5 mm in A0 –D0 .

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fibrous network with many pores and irregularly shaped hollows with raised edges and folds (Fig. 1C0 ). The ZP type III pores became bifurcated and occasionally conical as they penetrated the ZP. Zonae of ZP type IV had a very rough surface at low magnification (Fig. 1D) similar to ZP type III. At high magnification, however, many stringy filaments often covered and clogged the pores, making them difficult to discern (Fig. 1D0 ). These filaments made ZP type IV appear less compact. In Experiment 1, the frequency of ZP types for the highquality oocyte population showed that ZP type I was rare (2.7%; Table 1). In contrast, morphologies of ZP types II, III, and IV occurred in similar percentages (approximately onethird each) without significant differences among the frequency of these ZP types. The majority of dog donors (95.5%, n ¼ 42) produced an oocyte population that had either two (43.2%) or three (52.3%) ZP types. Of the 44 oocyte donors, only one (2.3%) had all of her oocytes classified as one ZP type, and only one (2.3%) had oocytes with all four ZP types. Oocyte diameter did not significantly differ among the four ZP groups (Table 2, Experiment 1). On the basis of examination of ZP type grouped by age of the oocyte donor, old dogs had at least three ZP types, and two age groups (juvenile, mature) had all four (Fig. 2). Two ZP types were found in oocytes from the prepubertal dog: ZP type II (n ¼ 2), ZP type III (n ¼ 1). When the frequency of oocytes with a ZP type I or II were pooled and compared with pooled oocytes with a ZP type III or IV for each dog age, the ZP type III and IV oocytes significantly increased with age and the ZP types I and II declined with age (P < 0.05). Moreover, juvenile dogs produced a significantly smaller proportion of oocytes with pooled ZP types III and IV than the older dogs (Fig. 2). Therefore, age of the dog donor affected ZP morphology. 3.2. Experiment 2: effect of oocyte viability on ZP structure As a next step in our study, we wanted to determine whether our methodology altered oocyte viability and possibly ZP surface ultrastructure from the time of collection from high-quality COCs and whether nonviable oocytes have a similar ZP to viable oocytes. Oocyte viability was evaluated with PI staining, in which oocytes with disrupted membrane integrity (dead) fluoresced red and viable oocytes did not (Fig. 3). After each oocyte was examined for viability and then processed for SEM, its

diameter was measured and ZP type assessed. In three of the eight dog oocyte donors, the average diameter of living oocytes (PI-negative) was significantly larger than that of their corresponding dead oocytes (PI-positive; Table 3). Oocytes from the other dogs were without significant differences. The average diameter of all of the living oocytes (PI-negative) was significantly larger than the dead oocyte population (PI-positive). The frequency of ZP types in living oocytes (PI-negative) had roughly equal distribution of ZP types II, III, and IV, but lacked ZP type I (Table 1, Experiment 2). Dead oocytes (PI-positive) had all four ZP types with the fewest oocytes having a type I ZP (5.3%) and a similar proportion of oocytes with ZP types II, III, and IV (Table 1). The overall frequency of ZP types in the live and dead oocyte populations did not significantly differ from each other. In Experiment 2, the living oocyte (PI-negative) diameters of the different ZP types did not significantly differ from each other (Table 2). In contrast, the average diameter of dead oocytes (PI-positive) with a ZP type IV was significantly larger than dead oocytes with ZP type II or III (Table 2). 3.3. Experiment 3: effect of COC quality on ZP structure In Experiment 2, ZP morphology was most likely not affected by the methods used to process oocytes from the time of collection from high-quality COCs. Therefore, we wanted to determine whether the variation observed in ZP morphology occurred before oocyte collection and thus was linked to COC morphology. To that end, oocytes were collected from both high-quality (grade 1) and low-quality (grade 2) COCs from four dogs and processed for SEM in Experiment 3. Average oocyte diameter did not significantly differ between high- and low-quality oocytes, although high-quality oocyte diameter in one dog was significantly larger than that of its corresponding lowquality oocytes (Table 4). When grouped by ZP type, the majority of the high-quality oocytes had a ZP type IV, but lacked a ZP type I (Table 1, Experiment 3). In contrast, the majority of the low-quality oocytes had a ZP type II and lacked the ZP type IV (Table 1). The distribution of ZP types in the high-quality oocytes used in Experiment 3 was not significantly different than the frequency of ZP types in the larger population of high-quality oocytes from Experiment 1 (Table 1). In contrast, the distribution of ZP types in low-quality oocytes was significantly different (Table 1). In

Table 1 Frequency (percentage) of ZP types of canine oocytes. Experiment

No. of oocytes

Zona pellucida Type I

Type II

Type III

Type IV

1 2

402

2.7 (N ¼ 11)

34.1 (N ¼ 137)

33.8 (N ¼ 136)

29.4 (N ¼ 118)a

Live Dead

47 57

0 (N ¼ 0) 5.3 (N ¼ 3)

29.8 (N ¼ 14) 35.1 (N ¼ 20)

29.8 (N ¼ 14) 29.8 (N ¼ 17)

40.4 (N ¼ 19)a 29.8 (N ¼ 17)a

High quality Low quality

35 34

0 (N ¼ 0) 2.9 (N ¼ 1)

31.4 (N ¼ 11) 79.4 (N ¼ 27)

20.0 (N ¼ 7) 17.6 (N ¼ 6)

48.6 (N ¼ 17)a 0 (N ¼ 0)b

3

Live, PI-negative oocytes; dead, PI-positive oocytes; high quality, oocytes isolated from high-quality (grade 1) COCs; low quality, oocytes isolated from lowquality (grade 2) COCs. a,b Different superscripts between rows indicate a difference (P < 0.05) in frequency of zona types per group.

M.O. Lunn, S.J. Wright / Theriogenology 80 (2013) 498–506

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Table 2 Diameter (mean  SEM) of canine oocytes according to ZP type. Experiment

No. of oocytes

1 2

402

Oocyte diameter (mm) with no. of oocytes for each ZP type ZP type I

ZP type II

ZP type III

ZP type IV

89.4  2.0 (N ¼ 11)

87.4  0.8 (N ¼ 137)

84.6  1.1 (N ¼ 136)

88.1  0.9 (N ¼ 118)

Live Dead

47 57

0 (N ¼ 0) 79.0  5.7 (N ¼ 3)

88.4  1.9 (N ¼ 14) 82.0  2.1a (N ¼ 20)

91.6  2.3 (N ¼ 14) 78.8  2.4a (N ¼ 17)

91.8  1.4 (N ¼ 19) 87.8  1.9b (N ¼ 17)

High quality Low quality

35 34

0 (N ¼ 0) 82.4 (N ¼ 1)

87.3  1.3 (N ¼ 11) 86.0  1.8 (N ¼ 27)

88.4  1.2 (N ¼ 7) 86.9 3.0 (N ¼ 6)

88.5  0.9 (N ¼ 17) 0 (N ¼ 0)

3

Live, PI-negative oocytes; dead, PI-positive oocytes; high quality, oocytes isolated from high-quality (grade 1) COCs; low quality, oocytes isolated from lowquality (grade 2) COCs. a,b Different superscripts within a row indicate differences (P < 0.05) between ZP types.

Table 2 (Experiment 3), the diameters of the high- and lowquality oocytes did not significantly vary for each ZP type in Experiment 3. 4. Discussion 4.1. Surface structure of the ZP The present study analyzed the morphology of the ZP external surface of ovarian canine oocytes with SEM. It was clear that the canine ZP was heterogeneous, with four distinct subtypes (ZP types I–IV). These data were consistent with the heterogeneous ZP structure reported for other mammals, including three ZP types in the goat [14], four in the pig and human [21,22], and four or five in the mouse [16,35]. There are contrasting opinions regarding the cause of ZP heterogeneity; some researchers have correlated ZP heterogeneity with oocyte nuclear maturation, whereas others have not. For example, a mesh-like spongy ZP structure was reported for mature human oocytes, whereas immature human oocytes had a compact,

100

Zona pellucida type (%)

90 80

Type I

70

Type II

60

Type III

50

Type IV

40 30 20 10 0 Juvenile Mature Old 6-11 mo 12-48 mo >48 mo Age of canine oocyte donor

Fig. 2. Relationship between ZP morphology and age of canine oocyte donor. The frequency of each ZP type for each age group is shown. The number of oocytes examined in the juvenile (n ¼ 14 dogs), mature (n ¼ 22 dogs), and old (n ¼ seven donors) age groups was 155, 184, and 60, respectively.

smooth surface [17]. However, other studies reported ZP heterogeneity in both immature and mature human oocytes, indicating no association of ZP type with oocyte nuclear maturation [21]. Similar results have been documented in mouse and pig oocytes [16,20,22,23]. Because a heterogeneous ZP surface morphology was reported for some species and others were unable to detect these variations in the same species, it has been suggested that the ultrastructural differences in the ZP surface could be due to variations in techniques used to prepare specimens for SEM [11,23]. Several researchers have pointed out that due to its extreme hydration state, the glycoprotein ZP is difficult to preserve for electron microscopy procedures, and care must be taken in preserving the ZP and in interpreting and comparing results [11,23]. In the present study, a graded series of ethanol with small increments starting with 50% ethanol was used to dehydrate oocytes for SEM [32]. All of the oocytes of the various groups in our study were processed for SEM using the same procedures. In addition, all oocytes from the same dog were processed for SEM together. Multiple ZP types were detected in oocytes collected from the same dog, and of 44 dogs, only one (2.3%) dog produced oocytes with only one ZP type. Moreover, the living (PI-negative) and dead (PI-positive) oocytes had similar frequency of ZP types. Therefore, we inferred that the techniques we used most likely did not affect ZP ultrastructure from the time of COC collection to viewing with SEM. For all of these reasons, we concluded that in our study, heterogeneity of the canine ZP surface was a physiological feature of the ZP and unlikely due to artifacts of specimen preparation. Alternatively, the similar frequency of ZP types may indicate that the zonae were affected in the same proportion. We confirmed the spongy appearance of the canine ZP surface previously reported [12,32,39,40]. It has been suggested that pores in the fibrous ZP network may be formed by transzonal processes of the granulosa cells that penetrate the ZP to contact the oocyte during oogenesis, and later, the spongy ZP surface becomes smooth as the transzonal processes retract during cumulus cell expansion [19,21,24]. Furthermore, canine ZPC/ZP3 and ZPB/ZP4 proteins were reported to be synthesized by the granulosa cells and ZPA/ZP2 made exclusively by the oocyte [8]. Thus, it is possible that the canine ZP proteins polymerized around the transzonal processes during oogenesis to form

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Fig. 3. Four canine oocytes stained with 10 mM PI to assess oocyte viability. (A) Fluorescence micrograph showing one PI-negative (viable; arrow) and three PI-positive (dead) oocytes. (B) Same oocytes imaged with brightfield microscopy. Oocytes are opaque in (B) due to their darkly pigmented, lipid-filled cytoplasm. Scale bar: 100 mm.

the zona’s three-dimensional fibrous meshwork. Our results showing a porous ZP surface were in accordance with these studies. Both the porous ZP meshwork and smooth ZP surface were observed in mouse, dog, pig, goat, cow, horse, and human oocytes [12,14–16,21,22,35,41]. In our study, the smooth and compact type of canine ZP (type I) was rare (2.3%). This ZP morphology was also rare (3.1%) in immature porcine oocytes [19,22]. In contrast, 15.6% of immature goat oocytes and 23.7% of immature mouse oocytes had the smooth ZP type [14,16]. That in our study ZP type I was absent from living (PI-negative) oocytes and rare or absent from high-quality COCs indicates ZP type I surrounds degenerating oocytes. This is in keeping with the previous studies, which showed the smooth ZP surface was observed in immature and atretic human oocytes [11,17], as well as degenerated and aged mouse oocytes [35] and degenerated canine oocytes [12]. This is in contrast to the studies that reported a smooth ZP was typically present on mature pig oocytes [22], which could be due to species differences. Because canine oocytes are typically ovulated at the GV stage, we expected grade 1 COCs to contain only GV oocytes. When Yamada et al. [42] examined the nuclear status of grade 1 oocytes from superovulated dogs, all oocytes were at the GV stage. However, others reported that whereas the Table 3 Diameter (mean  SEM) of canine oocytes in Experiment 2 according to oocyte viability. Dog

Live (PI-negative) Oocyte diameter (mm)

1 2 3 4 5 6 7 8 Total

80.8 89.6 79.4 86.8 93.5 99.5 98.1 94.9 90.7

        

1.4a 1.8a 0.5a 0.6a 0.9a 2.0a 0.9a 2.0a 1.0a

Dead (PI-positive) No. of oocytes

Oocyte diameter (mm)

4 5 4 12 3 3 11 5 47

80.8 85.0 70.0 88.4 92.5 72.2 96.6 85.3 82.6

        

5.1a 1.7a 1.3b 2.0a 0.5a 0.6b 1.8a 0.7b 1.3b

No. of oocytes 3 7 9 2 3 9 8 16 57

a,b Within a row, means without a common superscript letter differ (P < 0.05).

majority (73%-78%) of grade 1 oocytes recovered from ovaries of nonstimulated dogs were at the GV stage, 7% to 10% had resumed meiosis and 15% to 17% were degenerated [43,44]. Therefore, we inferred that up to 27% of the grade 1 oocytes in our population may not be at the GV stage; however, this does not account for all of the heterogeneity we observed in the ZP surface of high-quality canine oocytes. It could be related to cytoplasmic maturation events of the oocyte before nuclear maturation, although this would need to be directly tested. Taken together these results suggest the possibility that the variation in canine ZP surface morphology we observed was most likely not due strictly to oocyte nuclear maturation because this may account for only 7% to 10% of the oocyte population [43,44]. Studies using mouse, hamster, pig, or human oocytes did not report an association between appearance of the ZP surface and nuclear maturity of the oocyte [21–23]. However, other studies with mouse, goat, and human oocytes have reported changes in the outer ZP surface with oocyte nuclear maturation [14,16,17,20]. In addition, by using for their experiments, canine oocytes showing only ZP with multiple pores and hollows (our ZP type II), De los Reyes et al. [12] reported that the hole diameters enlarged after 72 to 96 hours of culture and concluded that the ZP surface was related to oocyte maturity in canines. Another peculiarity of canine reproductive biology is multioocyte (polyovular) follicles (MOFs), which contain 2 to 17 oocytes per follicle [8,45,46]. The prevalence of MOFs decreased with canine age and was 68.4% in dogs <1 year old and 14.3% in dogs aged 10 years [46]. That MOFs with more than two oocytes contained oocytes with different diameters and zona thickness suggests that they have different maturational stages. Perhaps zona surface morphology could also be affected in MOFs. An average 14% to 40% MOF frequency has been reported for sectioned canine ovaries [46,47]. Thus, up to 40% of the oocytes in the present study may have come from MOFs. Both viable and degenerating oocytes have been found in the same MOF [8,45,46], suggesting that canine ZP heterogeneity may be due to follicular atresia. Apoptosis plays a role in follicular atresia [48]. Lopes et al. [49] reported that the incidence of apoptosis in canine oocytes was low (5.1%), consistent with

M.O. Lunn, S.J. Wright / Theriogenology 80 (2013) 498–506 Table 4 Diameter (mean  SEM) of canine oocytes in Experiment 3 according to COC quality. Dog

High quality (grade 1) Oocyte diameter (mm)

A B C D Total

88.9 88.6 85.6 91.0 88.1

    

1.1a 0.9a 1.2a 1.3a 0.6a

Low quality (grade 2)

No. of oocytes

Oocyte diameter (mm)

10 11 10 4 35

85.8 90.6 88.5 80.9 86.1

    

2.3a 2.2a 0.9a 4.3b 1.5a

No. of oocytes 8 7 9 10 34

a,b Within a row, means without a common superscript letter differ (P < 0.05).

our smooth and compact ZP type I, suggesting that these surrounded degenerating oocytes in our study. In the present study, there were multiple ZP morphologies in oocytes collected from prepubertal, juvenile, and adult dogs. Due to the difficulty in collecting oocytes from pubertal dogs, our results from this age group were limited (one dog only), whereas data from the other age groups are based on 43 dogs (14 juvenile, 22 mature, 7 old). We reported that occurrence of ZP types I and II declined with age of the dog oocyte donor and types III and IV increased with donor age. Thus, we conclude that the quality of COCs varied with dog donor age. Whether age of dog donor affected meiotic competence in the canine as reported for the mouse is unclear [28]. Thus, we cannot speculate on the meiotic competence of oocytes with ZP types III and IV. However, one of the most critical events of fertilization is sperm binding to and penetration of the ZP [1,2]. Familiari et al. [11] reported that more human sperm bind a spongy ZP surface than a smooth one. Moreover, Palomino and De los Reyes [50] showed scanning electron micrographs of canine sperm interacting with a rough ZP type similar to our ZP types III and IV, which were prevalent in viable highquality oocytes. Taken together, these results suggest that ZP types III and IV represented high-quality, viable oocytes that may be capable of successful sperm interaction. 4.2. Oocyte diameter Canine oocytes collected from mature antral follicles were reported to have a larger diameter than those from preantral follicles, suggesting that larger oocytes were more mature [38,51,52]. Indeed, Hewitt and England [30] reported that oocytes with a larger diameter had greater meiotic competence. In the present study, that diameters of high-quality oocytes were similar among various ZP types (Table 2) were consistent with this. In addition, we observed older dogs had larger oocytes than younger dogs, and mature dogs had larger oocytes than juvenile dogs, indicating that age of the oocyte donor affected oocyte size (diameter). This was consistent with morphometric data of the canine ovary in which oocytes from secondary and tertiary follicles of adult dogs (>3 years) were larger than those of young dogs [51]. Moreover, in the present study, living (PI-positive) oocytes were significantly larger in diameter than dead (PI-positive) oocytes. Because PIpositive oocytes had compromised oolemmae, they would be more susceptible to shrinkage during SEM

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preparation. Funahasi et al. [18] reported that zona-intact pig oocytes shrank up to 50% during preparation for SEM; however, they used only short 50% and 100% ethanol dehydration steps, whereas we used a graded ethanol series with smaller increments, with each of longer duration, that should have reduced the amount of zona and oocyte shrinkage [32]. 4.3. Conclusions In conclusion, morphology of the ZP surface underwent dynamic structural rearrangements related to the viability of the oocyte and COC quality. We propose a model in which the ZP of mature canine oocytes is rough with numerous fenestrations and hollows and becomes smooth and amorphous with declining viability of the oocyte. In support of this, the frequency of ZP types in the different oocyte donor age groups had more ZP type III and IV oocytes with increasing oocyte donor age, and fewer oocytes with a ZP type II. Such changes in ZP morphology may be attributed to changes in the oocyte, and/or in the function and physical associations made by the adjacent cumulus cells. Our study has implications for canineassisted reproduction and there remains a compelling need to better understand the molecular mechanisms regulating ZP morphology and function to improve canineassisted reproduction, especially for endangered canids, and to develop highly effective contraceptives. Acknowledgments We thank Lynn E. Christy, D.V.M., and staff of the Northridge Veterinary Clinic for generously providing ovaries. We also thank David J. Wright, PhD, for help with image preparation and critical review of the manuscript. This research was funded in part by an American Kennel Club Canine Health Foundation Grant (#369) to S.J.W. and University of Dayton Graduate School Summer Fellowships to M.O.L. Author contributions: S.J.W. designed the experiments, supervised the project, analyzed the data, and finalized the manuscript. M.O.L. collected the ovaries, isolated and prepared the oocytes, viewed them with light and scanning electron microscopy, analyzed the data, performed statistical analysis, and drafted the manuscript. Both authors read and approved the final manuscript. Conflicts of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the reported research. References [1] Vajta G, Rienzi L, Bavister BD. Zona-free embryo culture: is it a viable option to improve pregnancy rates? Reprod Biomed Online 2010; 21:17–25. [2] Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD, editors. Physiology of reproduction. Raven: New York; 1994. p. 189–317. [3] Goudet G, Mugnier S, Callebaut I, Monget P. Phylogenetic analysis and identification of pseudogenes reveal a progressive loss of zona pellucida genes during evolution of vertebrates. Biol Reprod 2008; 78:796–806.

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