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Toxicology |
Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, fertilization, luteinizing hormone, ovulation, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Importantly, if the ovulatory surge of LH is blocked by pharmacological treatments during this critical period, it will typically occur on the following day [2, 3]. That is, the central nervous system mechanisms involved in the control of this event are linked to the circadian rhythm and are functional again 24 h later. Although a normal complement of ova are released 24 h later, the viability of the delayed oocyte is altered, as is the subsequent health and survivability of the ensuing embryo or fetus. For example, Butcher [4] reported a higher incidence of degenerating embryos following a 2-day delay in ovulation induced by sodium pentobarbital. Fugo and Butcher [5] noted that the smaller-than-normal Gestational Day 11 embryos were more likely to have chromosomal anomalies, and both Butcher [4] and Butcher et al. [6] noted an increase in fetuses with retarded development and in dead fetuses by Gestational Day 20. Although follicle growth continues after such a delay, the oocyte stays in meiotic arrest for an additional 48 h, and when meiotic development resumes, it is more rapid [7]. Ultrastructural studies of the released oocytes following this 2-day delay revealed the lack of a continuous layer of cortical granules lining the plasma membrane [8]. In addition, the mitochondria in the intrafollicularly aged oocyte were elongated with shelf-like cristae, suggestive of a higher metabolic state [8]. In models using estrogen-primed immature rats with a 48-h delay in ovulation produced by delaying hCG injection, the oocytes showed an increased rate of germinal vesicle breakdown and also showed a decreased ability to extrude the first polar body when matured in vitro. Once fertilized, an increase in supplementary sperm was observed [9].
Recent studies have examined the effect of environmental toxicants on the neuroendocrine regulation of the ovulatory surge of LH (for review, see [10, 11]). In particular, we found xenobiotics that alter norepinephrine (NE) and GnRH regulation of the LH surge can inhibit the ovulatory surge of LH if administered during the critical period of vaginal proestrus. These include noradrenergic-receptor blockers such as chlordimeform and amitraz [12]. Other examples include NE-synthesis inhibitors such as the dithiocarbamates (e.g., thiram and metham sodium [13, 14]), which suppress dopamine-ß-hydroxylase, and the chlorotriazines (e.g., atrazine) [15, 16].
In this earlier work, we reported that a single dose of thiram, a dithiocarbamate fungicide currently used for seed treatment, blocked ovulation at doses as low as 12 mg/kg when administered during the critical period for the neural trigger of the LH surge [14], with 50 mg/kg blocking the LH surge in 100% of females. We also found that although ovulation was delayed by 24 h in the treated females, the number of ova shed was not different from that in nondelayed females. However, when mated, the females in which ovulation was blocked with 50 mg/kg of thiram produced smaller litters, as evidenced by a significant reduction in the number of fetuses present on Gestational Day 20 [17]. Furthermore, although the number of implantation sites were slightly decreased on Gestational Days 7 and 11, most of the fetuses were resorbed after implantation. These effects on pregnancy outcome were similar to the results of our earlier study, in which litter size was reduced following a chlordimeform-induced delay in ovulation [18]. Thus, a single exposure to a toxicant affecting the NE mechanisms regulating pituitary LH secretion can delay ovulation and decrease embryo development and survival. Importantly, the reduced litter size that was found after a thiram-induced delay in ovulation was not attributable to a direct effect of the compound on the oocytes. Females dosed with thiram and bred the same evening experience a mating-induced LH surge, ovulate, and have normal-sized litters [17].
Therefore, we hypothesized that the reduced litter size in thiram-delayed rats is a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy (CLSM) to evaluate and characterize cortical granule (CG) localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal procedures were approved in advance by our facility's Institutional Animal Care and Use Committee. Ninety-day-old female Long-Evans hooded rats were obtained from Charles River Laboratories (Raleigh, NC) and housed in pairs in an AAALAC-accredited facility at 22°C on a 14L:10D photoperiod (lights-on at 0800 h). All animals were provided with food and water ad libitum. Following a 3-wk period of estrous cycle monitoring, only those females exhibiting at least three consecutive 4-day estrous cycles were used.
Dosing for Ovulatory Delay
Regularly cycling female rats were injected i.p. (0.2 ml/100 g body wt) with either control vehicle (0.5% methyl cellulose; Sigma Chemical, St. Louis, MO) or 50 mg/kg of thiram (98% purity; ChemService, West Chester, PA) in 0.5% methyl cellulose at 1245 h on the day of vaginal proestrus. This dosing regimen of thiram, as determined from our earlier work, interferes with the neural trigger of the LH surge and results in a 24-h delay of ovulation [14].
Fertilization
Groups of female rats were treated with thiram (50 mg/kg) at 1245 h on the day of vaginal proestrus and paired the following night (i.e., the evening of the presumed LH surge) with a proven breeder male. Control rats received vehicle at 1245 h on vaginal proestrus and were mated the same night. The female was then left with the male throughout the remainder of the night and was examined the next morning for the presence of a copulatory plug or sperm in the vaginal smear. The vaginal smear was also evaluated for cornification of vaginal epithelial cells to confirm estrus. At 1000 h on Day 0 (17 h after the LH surge) or on Day 1 (41 h after the LH surge), zygotes and 2-cell embryos, respectively, were collected and quantified according to the procedure described below for oviduct flushing.
Collection of Oocytes, Zygotes, and 2-Cell Embryos
Cumulus-enclosed mature oocytes, zygotes, and 2-cell embryos were flushed from the oviductal ampullae and counted [19]. If a cumulus mass was present, the oocytes were placed in 0.3% hyaluronidase in PBS (Gibco BRL, Gaithersburg, MD). Once the cumulus mass dispersed, the individual oocytes were washed with PBS, placed in droplets of PBS on a slide and mounted under 0.1-mm coverslips with Vaseline/paraffin posts, stained with acetolacmoid stain, and examined by phase-contrast microscopy. Pronuclei and associated sperm tails were counted in the zygotes. Supernumerary sperm, if present, were also counted. Some of the zygotes exhibiting supernumerary sperm were rolled under the coverslip before staining to confirm that the extra sperm were beneath the zona.
For the oocytes and zygotes that would be stained for subsequent cortical granule evaluation, Earles Balanced Salt Solution (EBSS; 25 mM, pH 7.3; Invitrogen, Rockville, MD) with 0.5% BSA (fraction V; Sigma) was used for flushing, and the oocytes were rinsed in EBSS without BSA. The zona pellucidae of the unfertilized oocytes were removed by placing them in a 0.1% pronase (Calbiochem, La Jolla, CA) solution of EBSS without BSA for 1–2 min. Zygote zonae are resistant to pronase at this stage, so zygotes were left zona-intact. The oocytes and zygotes were then rinsed in EBSS and placed in 3.0% paraformaldehyde for 30 min at room temperature and then overnight at 4°C until subsequent staining for cortical granule evaluation (see below). Fixative was made on the day of use by combining equal volumes of stock solutions (16% paraformaldehyde [Electron Microscopy Sciences, Fort Washington, PA] and double-strength buffered saline) followed by filtration (0.2 µm; GV-millex; Millipore Corp., Bedford, MA).
Staining Cortical Granules in Oocytes and Zygotes
Unfertilized, fixed oocytes were washed with the blocking solution (Dulbecco PBS [DPBS] with 0.1 M glycine and 0.3% BSA [pH]), permeabilized for 5 min in PBS containing 0.1% Triton X-100 with 0.3% BSA, and washed again. The lectin, Lens culinaris agglutinin (LCA), which is 
-D-mannose-specific in binding glycosylated molecules, was used as a CG probe as previously described [20, 21]. The oocytes were lectin-labeled according to a published protocol [22]. The oocytes were incubated with 10 µg/ml of LCA coupled to biotin (Polysciences, Warrington, PA, LcH#17627) in the wash solution (DPBS, 0.3% BSA, and 0.01% Triton X-100) for 30 min at room temperature, washed thoroughly, and subsequently incubated with 5 µg/ml of Texas Red-streptavidin (no. 9540SA; Gibco BRL). Chromatin was stained with 4',6'-diamidino-2-phenylindole (DAPI)/Hoechst 33258 (10 µg/ml) for 10 min. The oocytes were then mounted with Vectashield (Vector, RL-1000, Burlingame, CA) under 0.1-mm coverslips supported with Vaseline/paraffin posts and sealed with Pro Texx (Lerner Laboratories, Pittsburgh, PA).
In a procedure similar to that described above for lectin-labeling of cortical granules, the zygotes were incubated with 10 µg/ml of LCA conjugated to rhodamine (Vector, RL-1042) in wash solution. For CLSM, the samples were rinsed thoroughly and placed in depression slides (no. 30522-61; Edmund Scientific, Tonawanda, NY) filled with Vectashield/DAPI and sealed under 0.1-mm coverslips with Pro Texx.
Evaluation of Cortical Granule Distribution
Image acquisition using CLSM
The oocyte and zygote slides were imaged using a Leica CLSM (TCS-SP1; Heidelberg, Germany) that contained an inverted DMIRBE microscope and an Omnichrome laser emitting at three wavelengths (488, 568, and 647 nm). Excitation consisted of the 568-nm line for lectin staining and 365 nm for ultraviolet (UV) excitation for DAPI nucleic acid staining. A triple-dichroic beam splitter was used to collect UV (emission between 440 and 480 nm) and 568 (emission between 580 and 630 nm) emitted light. The emission pinhole aperture size was set to have the same diameter as that of the first airy ring of 151 µm. Each three-dimensional (3D) volume data set consisted of approximately 50 optical sections encompassing the entire depth (spacing, 
1.5 µm; depth, 75 µm) of the oocyte/zygote. The sample was first located with UV light using a 10x water-immersion objective, and then a parfocal 63x water-immersion lens (1.2 N.A.) was shifted into position to acquire the images. The oocyte or zygote containing the brightest fluorescence was measured first, and the following settings were kept constant during the CLSM scanning: laser power, photomultiplier tube (PMT) voltage, PMT offset, frame averaging, scanning speed, airy disk size, and step distances between adjacent sections. To minimize signal overlap between the UV and visible fluorescent lines, the images were sequentially scanned, first with the visible line (excited at 568 nm, emission at 580–630 nm) and then with the UV line (excited at 365 nm, emission at 440–480 nm). Bleaching was minimal (assessed with 3x averaging), and signal attenuation was minimal and consistent between control and delayed oocytes. The CLSM was evaluated to ensure that it was stable as described in two recent publications from our laboratory [23, 24].
Quantitation image analysis Imaris software (Bitplane, Zurich, Switzerland) was used to analyze the images of LCA-stained zygotes. This software produces 3D rotations and maximum projection images. The maximum projection images were used to visually score the zygotes based on distribution patterns of the cortical granule exudate.
Surpass software (Bitplane) is contained as a module and is used in conjunction with the 3D Imaris software to visualize 3D objects. Application of this software to the data set allowed for quantification of the number of fluorescent particles, surface area, and volume of the particles above user-defined specific threshold values. The Surpass program uses the marching cube algorithm that creates triangle models of constant-density surfaces. This method uses a "divide-and-conquer" approach to create interslice connectivity and create triangle topology, and it calculates triangle vertices using linear interpolation. Five different intensity thresholds (gray-scale values [GSV]) were defined and used to evaluate the data derived from the zygotes. These thresholds were determined by measuring the intensity of individual fluorescent particles and the neighboring area and then arbitrarily determining five acceptable GSV levels (100, 125, 150, 175, and 200). The data are dependent on the PMT voltage settings and can vary with different levels of intensity. Therefore, each group of treated zygotes was compared with controls evaluated the same day. By varying the size of triangles used to define particles, the smallest particles can be eliminated. In our case, the smallest particles (consisting of <50 triangles) were discriminated out of the analysis, with no limit at the upper end to be inclusive of all the aggregates observed. The particle number, area, and volume were then processed without using a smoothing algorithm. A similar procedure has been described to measure the volume and surface area of chondrocytes using geometric modeling of confocal serial sections [25].
Statistics
The proportions of oocytes with altered fertilization were analyzed using the Fisher exact test in GraphPad InStat (GraphPad Software, San Diego, CA). Unpaired Student t-tests were used to compare oocytes with subzona sperm between the delayed and the nondelayed controls. In both of the above analyses on fertilization, the sperm-positive females were the unit of measure. In the cortical granule analyses, the area, volume, and object numbers of the 3D Imaris images were also analyzed using unpaired t-tests with the individual zygotes as the unit of measure. The cortical granule exudate distribution patterns (type I, II, or III) between the control and the delayed zygotes were analyzed for trend using a chi-square distribution comparison.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The thiram dose of 50 mg/kg delayed ovulation for 24 h, as demonstrated in our earlier work. No differences were observed in the number or morphology of the mature oocytes between the nondelayed and the thiram-induced, 24-h-delayed oocytes at 14 h after the expected LH surge or shortly after ovulation. Staining with acetolacmoid stain revealed apparently normal oocytes that had reached the mature metaphase II stage and exhibited one polar body (data not shown).
Zygotes
A normal zygote was defined as an oocyte having one male and one female pronucleus, the second polar body, and a sperm tail inside. The thiram-induced, 24-h delay in ovulation resulted in a significant decrease in the percentage of normal zygotes 17 h following the presumed LH surge (Fig. 1). Oocytes without pronuclei were considered to be unfertilized, and oocytes with more than two pronuclei or more than one penetrated and decondensed sperm head, with associated sperm tails, were considered to be polyspermic. A significant increase was observed in the percentage of unfertilized zygotes in the thiram-delayed group (9.5%) as compared to the nondelayed group (1.1%) (Fig. 1). In addition, a significant portion of the zygotes in the delayed group (20.7%) were polyspermic when compared to controls (1.2%) (Figs. 1 and 2, A and B). Delayed zygotes also exhibited a 10-fold increase in the number of supplementary sperm as compared to the controls (Table 1 and Fig. 2). The location of these supplementary sperm in the delayed zygotes was determined to be within the perivitelline space, which was confirmed by rolling the zygotes under the coverslip before staining. As shown in Table 1, the mean number of supplementary sperm per delayed zygote was also significantly increased when compared to the nondelayed zygotes.
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2-Cell Embryos
A significant increase was observed in the number of unfertilized oocytes among 2-cell embryos (13.9% in delayed vs. 1.8% in controls), similar to that seen at the zygote stage. Likewise, a significant increase was observed in the percentage of 2-cell embryos with supplementary sperm (Table 1 and Fig. 2, C and D), and the mean number of supplementary sperm was significantly higher.
Cortical Granule Distribution and Analysis
No apparent differences were observed in the distribution or intensity of the fluorescence of LCA between the nondelayed and the delayed, unfertilized oocytes observed from 8 delayed and 11 nondelayed females (Fig. 3, A and B). The distribution pattern of the cortical granules were typical of that reported in mature oocytes [21], with a cortical granule-free domain over the metaphase II spindle. The chromatin also appeared normal.
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The fluorescent CG exudate on the zygote surface was classified into three distribution patterns, as illustrated in Figure 4: type I, CG exudate evenly dispersed on the surface of the vitelline membrane, either with a smooth appearance or as punctate individual particles; type II, CG exudate generally dispersed but with a few aggregates; and type III, CG exudate apparent as numerous aggregates on the surface. There also appeared to be differences in the intensity of the fluorescence between the aggregates and the individual particles, with the greatest intensity in the aggregates of distribution types II and III and the least intense fluorescence in the type I, evenly dispersed punctate particles.
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All the control zygotes exhibited staining patterns I or II (Fig. 5). In contrast, almost all the delayed zygotes exhibited staining pattern III (Fig. 5), with numerous aggregates. This trend was significant when examined by chi-square analysis (P < 0.0001). In addition, all the zygotes with polyspermy displayed a type III cortical granule exudate pattern.
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The three types of distribution patterns (types I, II, and III) were compared for number of particles of greater than 50 triangles (which would indicate aggregate size) above the 150 and 200 GSV thresholds (Fig. 6). At or above the 200 GSV intensity threshold, a significant difference was observed between the type I and type III groups and between the type III and type II groups, with the type III groups having a greater number of particles. However, at or above the 150 GSV intensity threshold, a significant difference was observed only in the number of particles between the type III (more numerous aggregates) and the type I (small evenly distributed) cortical granule distribution.
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To quantify these same types of intensity differences observed between the delayed and nondelayed zygotes, we determined particle size/aggregate and intensity. The mean number, surface area, and volume of the particles/aggregates differed between the control and the ovulatory-delayed zygotes on Day 0. Figure 7 shows the number of particles/aggregates that exceeded GSV intensity thresholds. A significant increase was observed in the number of bright aggregates in the delayed zygotes (i.e., those exceeding GSV thresholds of 150–200) compared to the controls. Figure 8 shows the total volume of particles/aggregates at different GSV thresholds. An increase was observed in the volume of the particles exceeding GSV thresholds of 100–200 in the delayed zygotes. In addition, a greater surface area was found in cortical granule particles from GSV levels of 125–200 in the delayed zygotes (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of supplementary sperm within the perivitelline space of the delayed zygotes clearly shows that the delayed oocytes failed to mount a normal block to polyspermy at the zona pellucida. However, whereas most (85%) of the delayed zygotes displayed supplementary sperm in the perivitelline space, only 21% were polyspermic. Thus, despite having a decreased protection against polyspermy at the level of the zona pellucida, the delayed oocytes in the present study appear to have another effective block to polyspermy at the level of the vitelline membrane. Such cell surface blocks to polyspermy have been noted in the rabbit [27].
The exocytosis of cortical granules in mammalian eggs is necessary for establishment of a block to polyspermy at the level of the zona pellucida [27]. Following fertilization, or sperm penetration and activation, a rise in intracellular free calcium [28] induces cortical granules to fuse with the plasma membrane and release their contents during the "cortical reaction." Some of these cortical granule components remain in the perivitelline space of fertilized mammalian oocytes to form a new extracellular matrix, called the cortical granule envelope, that persists during preimplantation development [29–32] and remains glycosylated, as revealed with LCA labeling [20]. Therefore, the establishment of this block to multiple sperm entry is required for normal mammalian development, and the timing of the cortical granule exocytosis appears to be important in this process. If it occurs prematurely, then fertilization failure may result. If it is delayed, asynchronous, or incomplete, then polyspermy may result.
Mammalian cortical granule components associate with the oocyte surface after fertilization, as revealed with LCA labeling of hamster oocytes. Following CLSM examination of the LCA-stained zygotes in the present study, we observed a significant difference in distribution and staining intensity between the control (types I and II) and the delayed (primarily type III) zygote, suggesting that cortical granule exocytosis or formation of the cortical granule envelope may be altered. The appearance of the delayed zygote exudate, with more numerous aggregates than the controls, seems to correspond to a more advanced stage of fertilization that has been observed previously in rat fertilized eggs [33]. For example, Raz et al. [33] showed that in fertilized eggs at the early stages of sperm decondensation, the CG exudate is visible as evenly distributed spots, whereas at later stages (i.e., when sperm decondensation was already completed or the pronuclear stage), cortical granule exudate appears as patchy aggregates scattered on the plasma membrane surface. Furthermore, although exocytosis is a very rapid process that follows the initial intracellular calcium rise, the dispersal of the CG material over the cell surface is relatively slow [34, 35]. Raz et al. [33] suggest that after the exocytotic process, rearrangement of the cortical granule exudate takes place on the plasma membrane, resulting in patches of cortical granule material. Based on these reports, we suggest that the increased aggregation (type III) in the delayed zygotes may be indicative of an advancement of this process, resulting in a less effective block to polyspermy at the level of the zona pellucida or oolemma.
Using the Surpass imaging software, the LCA staining was shown to be more intense in the delayed or type III group of zygotes, as indicated by the increased surface area, volume, and number of particles/aggregates defined by high-intensity thresholds (GSV 150–200). This is likely caused by clumping of the cortical granule exudate following the cortical reaction, making the fluorescence of the LCA more intense. It is generally understood that formation of cortical granules is a continuous process until the moment of ovulation, but exceptions have been noted in aging rabbit eggs that continue to accumulate cortical granules [36, 37]. Therefore, the delayed eggs may also have accumulated more cortical granules before fertilization and released more material on fertilization. Failure of the content of the cortical granules to disperse into the perivitelline space and an altered distribution of cortical granule exudate have also been reported following in vitro fertilization in porcine [38] and human in vitro fertilization [39].
Dandekar and Talbot [32] have shown that the cortical granule exudate in the perivitelline space may be important in blocking polyspermy in mammals, although only mouse, hamster, and human oocytes were examined. Delayed cortical granule exocytosis may result in the incomplete formation of the cortical granule envelope in the perivitelline space, thus resulting in polyspermic penetration into oocytes [32]. Wang et al. [38] also reported that a possible delay in cortical granule exocytosis may have resulted in polyspermy in the more mature porcine oocytes.
Mammalian eggs have a short postovulatory fertilizable life span. For example, immature and aged eggs have a higher incidence of abnormal fertilization or a temporal window of normal fertilizability (i.e., human in vitro fertilization studies). Changes in CG distribution during aging have also been observed in the eggs of mice and many other species and may increase the risk of polyspermy (for review, see [27]). In rats, Austin and Braden [26] found sharply increased rates of polyspermy following postovulatory delayed mating.
In summary, the present study demonstrated that exposure to a pesticide that delays the LH surge and ovulation also indirectly alters the time course of events in the oocyte that normally prevent polyspermy. This alteration results in polyspermy and, thereby, affects the outcome of pregnancy. Although we did not detect changes in cortical granules in the thiram-delayed, unfertilized egg, the distribution of the cortical granule exudate following sperm-egg interaction was altered, apparently allowing for polyspermy to occur in approximately 21% of the oocytes. It is also possible that other factors that are involved in activation of the oocyte on sperm-egg fusion are altered by the intrafollicular aging of the oocyte, thus allowing a disrupted cortical reaction and an alteration of the protective processes against polyspermy at the zona pellucida and plasma membrane of the oocyte.
The present study also suggests that such effects on cortical granule distribution, supernumerary sperm, and polyspermy can be generalizable to other environmental compounds that are able to suppress LH. Although differences exist in the ovarian cycle length of the rat and human, considerable homology is found between these two spontaneously ovulating species, both in the central nervous system-pituitary mechanisms controlling LH secretion and in the fertilization process. For example, some indirect evidence suggests that increased intrafollicular aging of human eggs may be correlated to an increased rate of polyspermy after in vitro fertilization [40]. Therefore, these observations in rat models may be applicable to humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Tammy E. Stoker, MD-72, Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. FAX: 919 541 5138; stoker.tammy{at}epa.gov
Received: 27 November 2002.
First decision: 12 December 2002.
Accepted: 14 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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