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Biology of Reproduction 60, 1384-1391 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Calcium Release and Subsequent Development Induced by Modification of Sulfhydryl Groups in Porcine Oocytes1

Zoltán Machátya, Wei-Hua Wanga, Billy N. Daya, and Randall S. Prather2,a

a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanism of Ca2+ release induced by modification of sulfhydryl groups and the subsequent activation of porcine oocytes were investigated. Thimerosal, a sulfhydryl-oxidizing compound, induced Ca2+ oscillation in matured oocytes. In thimerosal-preincubated oocytes, the amount of Ca2+ released after microinjection of inositol 1,4,5-trisphosphate (InsP3) or ryanodine increased strikingly, indicating that thimerosal potentiated both InsP3- and ryanodine-sensitive Ca2+ release pathways. Thimerosal also enhanced the sensitivity of oocytes to microinjected Ca2+ so that in pretreated oocytes a Ca2+ injection triggered a larger transient. Heparin at concentrations that normally block the InsP3-induced Ca2+ release were without effect; higher doses significantly increased the time leading up to the first spike. The thimerosal-induced Ca2+ release could not be blocked by procaine, and it did not require the formation of InsP3 since preinjection with neomycin did not prevent the oscillation.

Immunocytochemistry revealed that thimerosal treatment destroyed the meiotic spindle, preventing further development, an effect that could be reversed by dithiothreitol. The combined thimerosal/dithiothreitol treatment triggered second polar body extrusion in 50% of the oocytes, and as a result of this activation scheme ~15% of the in vitro- and ~60% of the in vivo-matured oocytes developed to blastocyst during a 7-day culture in vitro.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfhydryl groups are the most reactive groups on proteins, and their redox state can contribute to determining the structure and function of the protein [1]. Modification of these groups is known to affect many proteins involved in Ca2+ regulation and the operation of different ion channels: it alters the function of L-type Ca2+ channels [2], ATP-regulated K+ channels [3], and cardiac slow delayed rectifier channels [4]. Sulfhydryl reagents have also been shown to inhibit Ca2+-ATPases [5–7], stimulate the release of Ca2+ from sarcoplasmic reticulum [8], decrease the affinity of the inositol 1,4,5-trisphosphate (InsP3) receptor for InsP3 [7, 9], and decrease the number of InsP3 binding sites [7]. Thimerosal (sodium ethylmercurithiosalicylate), a hydrophilic sulfhydryl-modifying reagent, is able to oxidize sulfhydryl groups on proteins and bind to the protein through the resulting disulfide (S-S) bond.

It has also been reported that thimerosal is able to mobilize intracellularly stored Ca2+ in various cell types [10–12]. Moreover, it induces repetitive Ca2+ transients in hamster [13], mouse [14], rabbit [15], human [16], bovine [17], and porcine [18] oocytes. Its action appears to be mediated through oxidation of sulfhydryl groups on intracellular proteins because the effect can be completely inhibited with the sulfhydryl-reducing compound dithiothreitol (DTT). Since sulfhydryl groups on tubulin are also affected by thimerosal, and oxidation of tubulin sulfhydryl groups interferes with tubulin polymerization [19], oocytes treated with thimerosal cannot develop because of damaged meiotic spindles. This latter effect of thimerosal can also be reversed with DTT, and a combination of 200 µM thimerosal and 8 mM DTT treatment was reported to induce 42.0% of porcine oocytes to develop to the compact morula or blastocyst stage [18].

The mechanism underlying the actions of thimerosal in porcine oocytes is not known in detail. The objective of the present study was to address this question. Also, we wanted to investigate the changes that are induced in these cells by subsequent oxidation and reduction of sulfhydryl groups during the combined thimerosal/DTT treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocytes

Experiments were conducted according to institutional Animal Care Use Committee guidelines. All chemicals were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. Pig oocytes from ovaries collected at a slaughterhouse were matured in vitro as described previously [18]. Briefly, the follicular fluid from antral follicles was collected, and the cumulus-oocyte complexes were isolated. They were washed in Hepes-buffered Tyrode's medium containing 0.1% (w:v) polyvinyl alcohol (Hepes-TL-PVA) and matured in NCSU-23 medium supplemented with 10% porcine follicular fluid, 10 IU/ml eCG, 10 IU/ml hCG, 0.1 mg/ml cysteine, and 10 ng/ml epidermal growth factor. After being cultured for 22 h at 39°C under 5% CO2 in air, the oocytes were incubated in the same medium without hormonal supplementation for an additional 22 h. In one set of experiments in which the effect of the oocytes' age on the developmental potential was investigated, the culture without hormones lasted for either 20 h (early oocytes) or 28 h (aged oocytes). After maturation, the cumulus cells were removed from the oocytes by vigorous pipetting in the presence of 0.3 mg/ml hyaluronidase.

In vivo-matured oocytes were obtained from naturally cycling Large White gilts monitored for estrus once a day by exposure to a mature boar. Approximately 45 h after the detection of estrus, the gilts were anesthetized with approximately 18 mg/kg sodium thiopental followed by 2–5% halothane inhalation, their oviducts were flushed with 25 ml Hepes-TL-PVA, and the oocytes were collected. Both in vitro- and in vivo-matured oocytes were cultured in Hepes-TL-PVA until use.

Ca2+ Measurements

Fura-2 fluorescence was recorded using a Photoscan-2 photon counting fluorescence microscope system (Nikon Corp., Tokyo, Japan). Oocytes were incubated with 2 µM of the Ca2+ indicator dye fura-2 acetoxymethyl ester (AM) and 0.02% pluronic F-127 (both from Molecular Probes, Inc., Eugene, OR) in Hepes-TL-PVA for 40 min. After the dye was loaded, thimerosal was added to individual oocytes at a final concentration of 200 µM, and changes in the intracellular free Ca2+ concentration ([Ca2+]i) were measured. Fluorescent images were obtained by alternate excitation at 340 and 380 nm by means of a rotating chopper disk. The intensity of the emitted fluorescence was measured at 510 nm with a photomultiplier tube after background subtraction. [Ca2+]i levels are presented as fluorescence ratio values of the 340/380-nm excitation intensities; ratios of 1.2 and 6.5 correspond to approximately 65 nM and 602 nM Ca2+, respectively [20].

Microinjections

Fura 2-loaded oocytes were transferred into 50-µl drops of Ca2+- and Mg2+-free Hepes-TL-PVA supplemented with 100 µM EGTA. The dish was then placed onto a heated stage (39°C) of a Nikon Diaphot inverted microscope (Nikon Corp.). During microinjections, the oocytes were held in position with a fire-polished holding pipette. Microinjections were performed using a Narishige microinjector (Narishige Co. Ltd., Tokyo, Japan) as previously described [20]. The amount injected was approximately 40 pl (about 4% of the total cytoplasmic volume).

The following compounds were injected: D-myo-inositol 1,4,5-trisphosphate (InsP3), ryanodine, CaCl2, heparin (low molecular weight: sodium salt), de-N-sulfated heparin, and procaine. The carrier medium consisted of 10 mM Hepes and 100 µM EGTA (pH 7); concentrations of the injected compounds are shown as concentrations in the pipette.

Immunocytochemistry

Matured oocytes were treated with 200 µM thimerosal for 10–30 min; in certain cases, this was followed by a 10- to 30-min incubation with 8 mM DTT. Control groups also included nontreated oocytes and oocytes that were treated only with 8 mM DTT. The oocytes were then fixed in 3.7% paraformaldehyde in PBS for 2 h and permeabilized in PBS with 1% Triton X-100 detergent overnight at 4°C. Permeabilization was followed by a wash in PBS for 2 h at room temperature. Nonspecific binding of the antibody was blocked by a 30-min incubation in PBS containing 2% BSA and 150 mM glycine at room temperature. After the oocytes had been washed in PBS for 1 h, the microtubules were labeled by staining of the oocytes with 1 µg/ml fluorescein isothiocyanate-anti {alpha}-tubulin. It was followed by 3 washes in PBS with 1% Tween 20 (total washing time 2 h), and the DNA was stained using 5 µg/ml propidium iodide. The oocytes were then mounted in an antifade medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA) under posted coverslips. The slides were examined by laser-scanning confocal microscopy using a Bio-Rad (Hercules, CA) MRC-600 microscope equipped with a krypton-argon ion laser and mounted on an Optiphot II Nikon inverted microscope. The fluorescent images were recorded digitally and stored on a computer disk. Digital data were downloaded to a digital film recorder using Adobe Photoshop software (Adobe Systems, Inc., Mountain View, CA).

Chromatin Configuration and Development

After activation, the oocytes were cultured in Hepes-TL-PVA for 6 h, mounted under posted coverslips, and fixed in methanol:glacial acetic acid (3:1) for 48 h. Fixed oocytes were stained with 1% (w:v) aceto-orcein and evaluated for the presence of germinal vesicle, metaphase chromosomes, or pronuclei, using Hoffman Modulation Contrast Optics (Modulation Optics, Inc., Greenvale, NY) on an inverted Nikon Diaphot microscope at x400 magnification. Oocytes having one or more pronuclei were considered as activated. Some activated oocytes were incubated in NCSU-23 medium for 7 days; the culture medium was supplemented with 4 mg/ml BSA and 0.1 mg/ml cysteine [21]. At the end of the culture period, the number of oocytes that had developed to the blastocyst stage was recorded.

The developmental potential of in vivo-matured oocytes activated by sulfhydryl oxidation and reduction was also examined by culturing them in NCSU-23 medium supplemented with 4 mg/ml BSA and 0.1 mg/ml cysteine. Nontreated in vivo oocytes were used as controls. At the end of the incubation period, the rate of oocytes that developed to the blastocyst stage was determined in each group.

Statistical Analysis

Average data are presented as mean ± SEM. Percentage values of the experimental groups were tested for significance using chi-square analysis. The difference between the mean times required for the first Ca2+ transient to occur was determined by calculating protected least-significant differences using the multivariate General Linear Hypothesis of Systat [22].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ca2+ Measurements

Similar to what was reported earlier [18], the sulfhydryl-oxidizing agent thimerosal (200 µM) induced Ca2+ oscillation in 12 out of 12 porcine oocytes (12/12; Fig. 1). The first Ca2+ transient started 439 ± 23.0 sec after the addition of thimerosal, and the number of transients induced varied between 2 and 6. During the incubation with thimerosal, the basal [Ca2+]i gradually rose until individual spikes were no longer distinguishable.



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  		FIG. 1. Calcium oscillation in a representative porcine oocyte incubated in the presence of 200 µM thimerosal.

A short preincubation of the oocytes with thimerosal caused an increase in the amount of Ca2+ released after agonist stimulation from both the InsP3- and ryanodine-sensitive stores. Intracellular injection of 200 nM InsP3 triggered a small Ca2+ release in porcine oocytes (4/4; Fig. 2A). Pretreatment of the oocytes for 5 min with thimerosal (concentration as low as 30 µM) markedly potentiated the effect of InsP3, which subsequently induced a much higher Ca2+ transient (9/9; Fig. 2B). Thimerosal at this concentration induced a Ca2+ spike after > 10 min in 5 out of 5 oocytes (data not shown). Similarly, a low (50 µM) concentration of ryanodine, which is a ryanodine receptor agonist, induced only a negligible Ca2+ release (4/4; Fig. 2C). After a 5-min preincubation with 30 µM thimerosal, however, it evoked a large Ca2+ release, indicating that thimerosal potentiated the Ca2+ release induced by ryanodine (12/12; Fig. 2D).



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  		FIG. 2. Changes in the [Ca2+]i in porcine oocytes. The y-axis is the fluorescence ratio. A) 200 nM InsP3 induced only a small calcium transient. B) After a short preincubation with 30 µM thimerosal, the same InsP3 concentration triggered a huge spike. C) Similarly, low concentrations of ryanodine (50 µM in the pipette) stimulated a very small calcium release. D) The amount of calcium released as a result of the injection of 50 µM ryanodine increased strikingly after a 5-min preincubation in 30 µM thimerosal. E) The microinjection of 0.1 mM CaCl2 caused only slight changes in intracellular calcium levels; recurrent injections had the same effect. F) After a short thimerosal incubation (concentration 30 µM), the intracellular injection of 0.1 mM CaCl2 stimulated a large transient. Arrows indicate the time of microinjections.

The same effect of thimerosal on Ca2+ release was observed after Ca2+ injections as well. When 0.1 mM CaCl2 was microinjected into the oocytes, it evoked only a small Ca2+ transient; nor was the result of repetitive injections different (7/7; Fig. 2E). The same CaCl2 concentration triggered a much larger spike in oocytes that were preincubated with 30 µM thimerosal (9/9; Fig. 2F). Again, the increase in the [Ca2+]i elevation was probably due to Ca2+ release potentiated by thimerosal.

In order to determine which intracellular Ca2+ release channel receptor mediates the effects of thimerosal, we injected heparin into porcine oocytes. Heparin is known to be a competitive blocker of the InsP3 receptor [23]. Heparin at a concentration of 10 mg/ml was injected into the oocytes, and about 30 min after injection the oocytes were exposed to 200 µM thimerosal. At this concentration, heparin did not affect the Ca2+ oscillation induced by thimerosal in 6 of 6 oocytes. When 100 or 200 mg/ml heparin was injected into the cells, its effect was apparent in that it significantly (p < 0.001) prolonged the time leading up to the first Ca2+ transient in 9 of 15 oocytes (Fig. 3). In these oocytes, the first transient occurred 1318 ± 37.6 sec after the beginning of thimerosal treatment. The rest of the oocytes (6 of 15) did not show a Ca2+ spike during the 30-min measurement; in these cases, only a slow elevation of the intracellular Ca2+ level was detected. Control oocytes injected with 200 mg/ml de-N-sulfated heparin (the inactive form of heparin) showed no changes in their responses to thimerosal. When procaine (5 mM) was microinjected, it failed to block the Ca2+ transients triggered by thimerosal in 6 of 6 oocytes. An injection of 200 mM procaine was still without effect, while higher doses were detrimental to the oocytes (they induced coagulation of the cytoplasm and abnormally high baseline Ca2+ in 10 out of 10 oocytes; data not shown).



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  		FIG. 3. Representative recording of [Ca2+]i in a thimerosal-treated oocyte after microinjection of 100 mg/ml heparin. The transients in these oocytes were smaller and occurred considerably later.

Neomycin, the aminoglycoside antibiotic, was shown to prevent the formation of the second messenger InsP3 [24]. In the next experiment, we preinjected 10 mM neomycin into porcine oocytes, and 15–30 min later transferred them into 200 µM thimerosal. Neomycin preinjection could not inhibit the Ca2+ oscillation in 5 of 5 oocytes, indicating that the formation of new InsP3 is not a prerequisite for the thimerosal-induced Ca2+ release (data not shown).

Immunocytochemistry

In control oocytes, the microtubules were present in the meiotic spindle; the chromosomes were aligned on the metaphase plate that was located peripherally (Fig. 4A). Treatment of oocytes with thimerosal completely destroyed the meiotic spindle. No microtubules were visible in the oocytes (12 of 12) that were incubated with 200 µM thimerosal for 10 min (Fig. 4B). Similarly, no meiotic spindle was found in the oocytes treated with 200 µM thimerosal for 30 min, even after a 60-min incubation in the presence of the sulfhydryl-reducing agent DTT (Fig. 4C). However, when a 10-min incubation with thimerosal was followed by a 30-min culture in the presence of DTT, 14 of 14 oocytes had regenerated normal meiotic spindles. This combination of treatments had resulted in 73.8% pronuclear formation previously [18]. By the end of the DTT incubation, some oocytes (6 out of 14) were already at the anaphase stage (Fig. 4D). DTT treatment alone did not affect the nuclear status of the oocytes: these cells remained arrested at second metaphase and displayed normal metaphase plates after either a 30-min or a 120-min treatment with DTT (Fig. 4, E and F).



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  		FIG. 4. Immunofluorescent localization of microtubules in porcine oocytes. Green and red images display microtubules and chromatin, respectively. A) In control oocytes, the microtubules were found in the meiotic spindle. B) No meiotic spindle was visible in oocytes that were incubated with 200 µM thimerosal for 10 min. C) The meiotic spindle was not observed in the oocytes treated with thimerosal for 30 min even after a 60-min treatment in the presence of DTT. D) In the oocytes that were treated with thimerosal for 10 min, the meiotic spindle was regenerated by a 30-min incubation with DTT. Some of these oocytes reached anaphase by the time of fixation. E) A 30-min or F) a 120-min DTT treatment had no effect on the microtubules. Bar represents 30 µm.

Chromatin Configuration and In Vitro Development

In order to characterize the effects triggered by sulfhydryl oxidation and reduction, oocytes treated with thimerosal/DTT were fixed 6 h after treatment and stained for chromatin configuration. This revealed that 50% (91 of 182) of the early (42 h old) oocytes treated with thimerosal/DTT extruded their second polar bodies, while second polar bodies were visible only in 35.3% (47 of 133) of the aged (50-h-old) oocytes (Table 1). Control oocytes that had been activated by an electrical pulse showed second polar body extrusion in only 15.4% (early) and 17% (aged) of the cases.


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  		TABLE 1. Effects of the combined thimerosal/DTT treatment and electrical stimulation on pig oocytes.

Pronuclear formation was highest in the aged oocytes activated by electroporation (100%); thimerosal/DTT-activated early and aged oocytes showed 70.3% (128 of 182) and 82.7% (110 of 133) pronuclear formation, respectively. The rate of oocytes that developed to the blastocyst stage was around 15% in each experimental group.

Developmental Potential of In Vivo Oocytes

The 10-min thimerosal/30-min DTT treatment induced development of the activated oocytes to the blastocyst stage ([18] and present results). In the next set of experiments, the developmental potential of the in vivo-matured oocytes activated by this protocol was investigated. From the oviducts of the three donor gilts, 42 in vivo-matured oocytes were collected, 32 of which were treated with thimerosal/DTT and 10 of which were used as negative controls. After thimerosal/DTT stimulation and a 7-day culture in NCSU-23 medium, 59.4% (19 of 32) of the oocytes formed blastocysts, while none of the control oocytes developed to this stage (p < 0.001). The average cell number of the blastocysts determined by Hoechst 33342 staining was 19.7 ± 3.5.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Consistent with earlier observations, thimerosal induced Ca2+ oscillation in porcine oocytes. The oscillation began with a large Ca2+ transient within 10 min, which was followed by some additional smaller spikes. A characteristic feature of the action of thimerosal was the gradual elevation of the basal [Ca2+]i. Thimerosal reportedly inhibits Ca2+-ATPases and thus prevents the removal of excess Ca2+ from the cytosol [5–7]. The sustained increase in [Ca2+]i was probably attributable to the inhibition of these pumps.

Several theories have been proposed to explain the mechanism by which thimerosal induces Ca2+ release from intracellular stores. According to one hypothesis, thimerosal affects the binding of the stored Ca2+ to Ca2+-binding proteins by lowering the affinity and/or the capacity of the binding proteins for Ca2+ [25]. In sea urchin egg homogenates, thimerosal had its potentiating effect on two distinct Ca2+-releasing channels and caused a continuous leak of sequestered Ca2+. It was suggested that thimerosal increases the concentration of intravesicular free Ca2+ and thus triggers the continuous leak of Ca2+ until a new equilibrium state is reached across the endoplasmic reticulum membrane [26, 27]. A sarcoplasmic reticulum (SR) protein called triadin was characterized as a protein having a possible disulfide-bond site, and it was proposed to interact with the intravesicular Ca2+-binding protein calsequestrin [28, 29]. Although the presence of triadin has not been shown in sea urchin eggs, these proteins are thought to be potential targets in the action of thimerosal to potentiate Ca2+ release.

Another model postulates that sulfhydryl reagents directly open and close Ca2+ release channels by oxidizing and reducing critical sulfhydryl groups on the Ca2+ release protein [30]. It was reported that oxidizing agents caused Ca2+ release from SR vesicles and that they bound to a 106-kDa protein that had Ca2+ channel activity in a reconstituted system [8, 31]. However, the mechanism by which oxidation of channel proteins directly causes opening of channels has not been demonstrated.

It has also been hypothesized that the mechanism of the thimerosal-induced elevation in the [Ca2+]i is due to the sensitization of the InsP3 receptor by thimerosal to basal InsP3 levels [12, 32–34]. To induce a half-maximal mobilization of Ca2+ stores in HeLa cells, a 2.6-fold lower InsP3 concentration was sufficient after a preincubation with thimerosal [12]. The present data indicate, however, that a pretreatment with thimerosal increased the Ca2+ flux through not only the InsP3 but also the ryanodine receptor channels, suggesting that thimerosal has its effect on both receptor types in porcine oocytes. This is consistent with earlier findings that in skeletal muscle thimerosal interacted with ryanodine receptors [35] and in sea urchin eggs it potentiated both InsP3-sensitive and -insensitive Ca2+ release pathways [25].

In hamster eggs, thimerosal reportedly decreased the amount of injected Ca2+ necessary to generate a Ca2+ spike [13, 33]. Regenerative and propagating Ca2+ release was induced when [Ca2+]i was elevated to about 300 nM by microinjection. Since hamster oocytes do not have ryanodine receptors [36] through which the classical Ca2+-induced Ca2+ release (CICR) could operate [37], the microinjected Ca2+ apparently sensitized the InsP3-induced Ca2+ release (IICR) mechanism, and Ca2+ release was triggered by endogenous InsP3 levels. Thimerosal was found to lower the threshold level of Ca2+ by further sensitizing Ca2+-sensitized InsP3-induced Ca2+ release (CSIICR, [37, 38]). Microinjected Ca2+ also triggered a much larger Ca2+ transient in thimerosal-pretreated porcine oocytes. The situation in the porcine oocyte is more complex, however, as porcine oocytes were shown to have both InsP3- and ryanodine-sensitive Ca2+ pools [20]. To answer the question whether the increase in the Ca2+ release is the result of a CICR through the sensitization of the ryanodine receptors or a CSIICR via the sensitization of the InsP3 receptors needs further investigation. However, the fact that the ryanodine receptor antagonist procaine had no effect on the Ca2+ release induced by thimerosal suggests the involvement of the InsP3 receptors rather than the ryanodine receptors.

Heparin concentrations that could totally inhibit a Ca2+ release induced by InsP3 in bovine and porcine oocytes [20, 39] proved to be ineffective in blocking the effects of thimerosal. High concentrations of heparin could significantly increase the time leading up to the first spike, although some oocytes showed no Ca2+ transients during the 30-min measurement. In these cases, a continuous elevation of the baseline Ca2+ level was observed, possibly indicating a slow but continuous leaking of Ca2+ from the stores. Low concentrations of heparin were without effect in blocking the Ca2+ oscillation stimulated by thimerosal in mouse oocytes as well [40], while high doses blocked the thimerosal-evoked calcium spikes in rabbit and bovine oocytes [15, 17]. One possible explanation for this high-concentration requirement might be that, as suggested earlier [12], thimerosal increases the sensitivity of the InsP3 receptor to InsP3. Since heparin is a competitive agonist of the InsP3 receptor, its effectiveness in blocking the receptor, whose affinity for InsP3 was increased by thimerosal, is reduced. In addition, thimerosal can potentiate the ryanodine receptors that might also be involved in the Ca2+ release, although the attempts to block the oscillation with procaine remained futile. In hamster oocytes, a monoclonal antibody against the InsP3 receptor totally blocked such spiking [33].

Neomycin did not affect the release of Ca2+ in response to thimerosal. Neomycin was shown to prevent the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP2) and thus the production of InsP3 in sea urchin egg plasma membrane [24]. It also abolished the wave of Ca2+ release in sea urchin eggs [41] and was used to inhibit receptor-stimulated inositol phosphate formation in rat chromaffin cells [42]. The finding that thimerosal is able to induce Ca2+ release without stimulating the production of InsP3 supports earlier reports in which thimerosal inhibited the production of inositol phosphates in HeLa cells [12].

Immunocytochemistry showed that, as in the mouse [43], sulfhydryl oxidation completely damaged the meiotic spindle in porcine oocytes. This seems to be the reason why mouse and porcine oocytes did not develop after a treatment with thimerosal alone [18, 43]. Reduction of sulfhydryl groups with DTT, however, could reverse this damage. Although long (30-min) thimerosal incubations permanently destroyed the microtubules, the detrimental effects of shorter treatments (10 min) could be reversed. After being released from the meiotic arrest by the Ca2+ release induced by thimerosal, in many cases the meiotic division progressed to anaphase by less than an hour post-activation, demonstrating that the spindle in these oocytes was fully functional.

The combined oxidation and reduction of sulfhydryl groups is very effective in inducing the resumption of meiosis and supporting embryonic development. After completing the second meiotic division, a relatively high percentage of oocytes extruded their second polar bodies. Second polar body extrusion, which is a general phenomenon after fertilization, does not always happen after parthenogenetic activation [44]; it occurred in only 17% of the electroporated oocytes. The fact that up to 50% of the thimerosal/DTT-treated oocytes possessed a second polar body might indicate that the activation triggered by sulfhydryl modification is closer to physiological conditions than electroporation. Although aged oocytes normally activate more effectively [45, 46], this was not an advantage in the case of thimerosal/DTT activation: aged oocytes activated this way formed pronuclei at about the same rate as younger ones. The effectiveness of activation by sulfhydryl modification is further supported by developmental data obtained using in vivo oocytes. While approximately 15% of in vitro-matured oocytes developed to the blastocyst stage, the results indicate that this rate in the case of in vivo-matured oocytes is considerably higher.

We conclude that thimerosal, the sulfhydryl-oxidizing agent, potentiates two Ca2+ release pathways and induces Ca2+ oscillation in porcine oocytes. Without the involvement of newly synthesized InsP3, it sensitizes the Ca2+ release channel receptors to InsP3, ryanodine, and Ca2+ so that these agonists are capable of triggering the release of more Ca2+ from the stores. Thimerosal also oxidizes sulfhydryl groups on tubulin, thus destroying the meiotic spindle and preventing the formation of pronuclei. In a combined treatment with the reducing compound DTT, however, it is able to induce activation and development to the blastocyst stage in a high percentage of oocytes.


   FOOTNOTES
 
1 This material is based upon work supported by the Food for the 21st Century and the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, under agreement No. 95-37203-2073. The manuscript is a contribution from the Missouri Agriculture Experiment Station Journal Series No. 12,857. Back

2 Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211. FAX: 573 882 6827; randall__prather{at}muccmail.missouri.edu Back

Accepted: January 26, 1999.

Received: December 3, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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