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BOR - Papers in Press, published online ahead of print April 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038307
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BIOLOGY OF REPRODUCTION 73, 308–314 (2005)
DOI: 10.1095/biolreprod.104.038307
© 2005 by the Society for the Study of Reproduction, Inc.

Role of Glutathione in Reproductive Tract Secretions on Mouse Preimplantation Embryo Development1

James J. Salmen , Frank Skufca 2 , Ani Matt , Gene Gushansky , Amy Mason , and Catherine S. Gardiner 

University of Northern Colorado, Department of Biological Sciences, Greeley, Colorado 80639


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the hypothesis that glutathione (GSH) in reproductive tract secretions (RTS) protects the preimplantation embryo from endogenous reactive oxygen species and is important for normal development during the embryo's sensitive period when it is incapable of synthesizing GSH de novo. Mice were administered buthionine sulfoximine (BSO) to inhibit GSH synthesis and decrease GSH concentration in RTS. Embryos were then allowed to develop either in vivo or in vitro in the presence of RTS and the GSH concentration of the embryos was quantified by HPLC and embryonic development was recorded. GSH concentration in RTS did not differ over the phases of the estrous cycle, but there were significant decreases in GSH concentration on Day 2 of gestation and due to BSO treatment. Embryos allowed to develop in vivo and in vitro in RTS with decreased GSH concentration did not exhibit decreased development or GSH concentration. Oocytes exposed to BSO during maturation in vivo experienced a significant decrease in GSH concentration and an increase in percent of degenerate embryos when compared with control. These data suggest that most of the GSH in RTS does not play a critical role in normal preimplantation embryo development but that GSH stored in the oocyte during maturation has an important role in subsequent embryo development. Our studies do not exclude the possibility that GSH in RTS plays an important role in protection of the preimplantation embryo during exposure to some toxicants.

early development, embryo, female reproductive tract, oviduct, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione in the reduced form is a tripeptide (GSH, {gamma}-glutamylcysteinylglycine) that is used as a storage and transport form of cysteine for cells and functions to protect cells from toxicants and reactive oxygen species (ROS) that originate from exogenous and endogenous sources [1]. Synthesis of GSH de novo is a two-step process, which requires glutamate-cysteine ligase (GCL) and glutathione synthetase. GCL catalyzes the rate-limiting step and is inhibited by buthionine sulfoximine (BSO) [2].

GSH has an important role in the preparation of the oocyte and initiation of embryo development. An increase in the GSH concentration of the oocyte is associated with ovulation and is required for progression of embryo development after fertilization in vitro [3, 4]. It has been shown that a decrease in oocyte GSH concentration before in vitro fertilization prevents pronucleus formation and blocks development of the embryo [5, 6]. In vitro, supplementation of oocyte culture media with cysteine, a rate-limiting substrate in GSH synthesis, increases the intracellular GSH concentration of the oocyte and promotes pronuclear formation [7].

The preimplantation embryo is sensitive to toxicants and ROS and will not develop successfully if it is exposed to such environmental stresses. It has previously been demonstrated in mice that, after fertilization, the GSH concentration of embryos decreases by 90% from the oocyte to the blastocyst stage in vivo [8]. This is due at least in part to the cleavage-stage embryo's inability to synthesize GSH de novo [9]. This inability to synthesize GSH de novo leads to decreased embryonic development in vitro due to exposure to toxicants or oxidative stress [9, 10]. The preimplantation embryo is sensitive to very low levels of chemicals that cause GSH oxidation in vitro, such as tertiary-butyl hydroperoxide [8]. The cleavage-stage embryo is also very sensitive to chemicals that cause GSH depletion, such as diethyl maleate in vitro [9] compared with the postimplantation embryo [11].

Interestingly, in vivo exposure of embryos to acetaminophen, another GSH-depleting agent, does not cause a significant decrease in the percentage of embryos developing to the blastocyst stage [12]. In culture, however, cleavage-stage embryos exposed to acetaminophen exhibit a dose-dependent decrease in the percentage of embryos developing to the blastocyst stage [12]. Taken together, these data provide evidence of a maternal mechanism for the protection of the embryo during the preimplantation phase of development.

Extracellular GSH is present in reproductive tract secretions (RTS) and may provide protection for the sensitive cleavage-stage embryo [13]. GSH is found in extracellular fluids such as bile, alveolar lining fluid, cerebrospinal fluid, plasma, saliva, sweat, seminal fluid, and milk [14], and its concentration can be increased to provide additional protection in response to stress [15]. One method of protection provided by extracellular GSH is through intact uptake of extracellular GSH for use within the cell, as is the case in the renal tubules [16, 17] and intestines [18]. Intact uptake of GSH in the renal tubules contributes more to the intracellular GSH concentration than does de novo synthesis [16, 17]. Another mechanism whereby extracellular GSH affords protection for cells is the extracellular conjugation of GSH with toxicants, preventing the toxicants from entering the cell and causing damage. Glutathione transferase in the mucus of the rat small intestine extracellularly catalyzes the conjugation of xenobiotics with GSH provided by bile [19]. In bovine pulmonary artery endothelial cells, extracellular GSH conjoins with oxidant-inducing chemicals, inhibiting their ability to create ROS [20]. Extracellular protection from oxidation is also provided by GSH as a substrate for extracellular glutathione peroxidase, an enzyme responsible for the reduction of hydroperoxides. Extracellular glutathione peroxidase is a class of glutathione peroxidase that functions extracellularly and includes the products of several genes. Extracellular glutathione peroxidase is found on many extracellular borderlines and exhibits high levels of activity in sites such as amniotic fluid [21, 22]. In addition, extracellular glutathione peroxidase activity modulates extracellular signaling factors and protein redox status [21]. Extracellular GSH is important for the maintenance of membrane redox status and protection of surface proteins. A 50% decrease in extracellular GSH results in an 85% decrease in surface sulfhydryl groups of peripheral blood mononuclear cells [23]. These studies suggest that GSH in extracellular fluid may function to neutralize toxicants and reactive oxygen species, provide a source for intracellular GSH through uptake, and/or modulate the redox status of membrane proteins to protect the cleavage-stage embryo.

We hypothesize that GSH in RTS protects preimplantation embryos from toxicants and endogenous reactive oxygen species during the period of development when they are unable to synthesize GSH de novo. To begin to test this hypothesis, we measured GSH concentration in mouse RTS during the estrous cycle and over the period of gestation when the embryo is unable to synthesize GSH de novo. We also examined the effect of decreasing the concentration of GSH in the RTS on embryo development in vivo and in vitro.


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

NSA mice were originally purchased from Harlan (Indianapolis, IN) and subsequently bred in our facility. All experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals [24]. Chemicals were purchased from Sigma-Aldrich (http://www.sigmaaldrich.com), except for Bradford reagent, which was acquired from Bio-Rad (www.bio-rad.com).

Determination of the Stage of the Estrous Cycle by Vaginal Cytology

The stage of the estrous cycle was determined by microscopically evaluating vaginal cytology [25] after staining of collected vaginal epithelial cells with Harris modified hematoxylin solution (Sigma cat. no. HHS-128) and eosin Y solution (Sigma Cat. No. HT110-1-128). The results were recorded and mice were grouped according to phase of the estrous cycle.

Collection of Reproductive Tract Secretions for Quantification of GSH

Mice, in different phases of the estrous cycle or gestation, were killed by cervical dislocation and prepared for collection of RTS. An incision was made and the reproductive tract was gently lifted and supported by forceps, separating it from the other organs. The reproductive tract was rinsed with flushing buffer containing 0.9% NaCl, 50 mM HEPES, and 2 mM diethylenetriaminepentaacetic acid (pH 7.4). Uterine secretions were collected by flushing the uteri with flushing buffer after the uterus had been tied off at the cervix to prevent buffer from flowing into the vagina. Eighty microliters of flushing buffer was injected into the lumen of the uterus while the reproductive tract was still intact within the mouse and subsequently collected via a 30-gauge needle on a 1-ml syringe. For collection of oviduct secretions, the reproductive tract was removed from the mouse and transferred to a watch glass. The string, still tied to the uterus, was used to suspend the cut end of the uterus and prevent it from contacting and contaminating the watch glass while the oviduct was being flushed. A 30-gauge needle was inserted into the uterotubal junction and the oviduct was flushed with buffer. The reproductive tract flushing was collected from the dish and placed into a centrifuge tube. The samples were centrifuged at 13 000 x g, for 10 min to remove cellular debris. Fifty microliters of the supernatant was immediately derivatized for quantification of GSH and oxidized glutathione (GSSG) using the protocol described below. Samples were analyzed by HPLC the following day. Five microliters were analyzed for protein concentration using a modification of the Bradford method [26]. To compensate for possible differences in the size of the reproductive tract or the volume of secretion affecting the concentration of GSH, GSH is expressed in nmol GSH/mg protein in the flushing. GSH concentrations in RTS were also calculated as molar concentrations, and the results of the statistical analysis of treatment effects were the same as the analysis expressed as nmol GSH/mg protein.

Superovulation and Embryo Collection

For in vivo preimplantation development, pubertal NSA female mice were maintained on a 14L:10D cycle. The mice were synchronized and superovulated by intraperitoneal (i.p.) injection of 10 IU equine chrorionic gonadotropin (eCG) followed 44–48 h later by 5 IU of human chrorionic gonadotropin (hCG). Females were then mated with proven breeder males and copulation was determined by vaginal plug detection. The day of vaginal plug detection was designated as Day 0 of gestation. For collection of oocytes, females were not bred and were instead killed the day following hCG injection and the oviducts were removed and flushed. Bred females were killed on either Day 0, 1, 2, or 3 of gestation. For collection of two-cell stage embryos, the oviducts were removed and flushed on Day 1. For four-cell- to eight-cell-stage embryos, both the oviducts and the uteri were removed and flushed on Day 2. For collection of blastocysts, the uteri were removed and flushed on Day 3. All collections took place during the afternoon, beginning at approximately the midpoint of the 14-h light period. Embryos were collected by flushing oviducts or uteri with M16 culture medium [27]. Embryos were microscopically evaluated and indication of fertilization, stage of development, quality of embryos, and abnormal features were recorded.

Administration of BSO In Vivo

To examine the effect of a decrease in the concentration of GSH in RTS, mice were treated with BSO to block GSH synthesis. Mice were placed into either control or BSO-treatment groups. Mice in the BSO groups received i.p. injections of 0.2 M BSO (4 mmol/kg body weight/ injection) in 0.1 M NaCl four times per day at 3-h intervals, starting at 0900, following the previously published protocol [5]. Mice were treated with BSO from Day 0 until embryo or reproductive tract flushing collection or from 60 h before ovulation until injection of hCG. Mice in the control group received sham injections of 0.1 M NaCl on the same schedule.

Collection of Reproductive Tract Flushings for Embryo Culture In Vitro

Mice were killed and RTS were collected by flushing the oviducts or uteri with 80 µl of M16 as described above. For Day 1 RTS collection, oviductal flushings were collected from 5–10 mice and pooled together to provide enough flushings for culture medium. For Day 2 and 3 collections, the uteri of 5–10 mice were flushed and again pooled by day of gestation. After collection, reproductive tract flushings were centrifuged at 13 000 x g for 10 min. When centrifugation was completed, the samples were filter sterilized using 0.22-µm centrifugation filters and added to culture medium for immediate use.

Culture of Embryos in Media with Reproductive Tract Flushing

Reproductive tract flushings collected on Days 1, 2, or 3 from control and BSO-treated mice were used to supplement M16 culture medium at a 1:1 ratio with BSA added to a final concentration of 4 mg BSA/ml in all culture media. Embryos were collected by flushing the oviducts of mice during the afternoon of Day 1 of gestation, beginning at the midpoint of the light cycle, with M16 culture medium into a watch glass. The embryos were collected, pooled, and subsequently washed through a minimum of five 50-µl drops of culture medium. Embryos were randomly divided into groups of 10, and 10 embryos were placed into each 10-µl drop of the various culture media under mineral oil and placed into a humidified 37°C, 5% CO2 in air incubator. Experiments used a total of 160 embryos each in Day 1 control, Day 1 reproductive tract flushing, and Day 1 BSO reproductive tract flushing. Embryos were moved from culture drops containing Day 1 reproductive tract flushing to culture drops containing Day 2 reproductive tract flushing and finally to drops containing Day 3 reproductive tract flushing to match the stage of embryo development with the physiological status of the reproductive tract. Controls were also transferred to new culture drops on the same schedule. Embryos were microscopically evaluated and quality and stage of development were recorded daily.

HPLC Detection of GSH and GSSG

Measurement of GSH and GSSG in reproductive tract flushings and embryos was conducted by HPLC using an aminopropyl silica column with a methanol-sodium acetate gradient system [28] and using fluorescent labeling of GSH and GSSG with dansyl chloride [29]. The amount of glutathione in reproductive tract flushings and embryos was determined by comparison of the resulting peak area to the peak area of the internal standard ({gamma}-glutamylglutamate) and to peak areas in standard curves. GSSG concentration in samples was typically below the threshold of detection. Data are expressed as nmol/mg protein for reproductive tract flushings and pmol/embryo or oocyte.

Statistical Analysis

Differences in percentages of morphological development of embryos were determined using arcsine transformation, ANOVA, and Fisher least-significant-difference procedures. Differences in GSH concentrations were determined using ANOVA and Fisher least-significant-difference procedures. Glutathione values represent means ± SEM and each value represents a minimum of three replications with at least 30 embryos for each measurement. Measurements for reproductive tract flushings were conducted a minimum of three times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GSH Concentrations in Reproductive Tract Flushings

GSH concentrations in reproductive tract flushings were measured over the stages of the estrous cycle and the first 3 days of gestation (Figs. 1 and 2, respectively). GSH was also measured in reproductive tract flushings for the first 3 days of gestation during treatment with BSO to inhibit GSH synthesis. Over the stages of the estrous cycle, the GSH levels were not significantly different within the oviduct or uterus (Fig. 1). However, over the days of gestation, the levels of GSH do differ significantly due to both treatment with BSO and day of gestation. GSH was lower on Day 2 (d2) of gestation than on Day 1 (d1) or Day 3 (d3), and GSH was lower on every day of gestation due to treatment with BSO (Fig. 2).



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  		FIG. 1. GSH concentration of reproductive tract flushing collected midday during various stages of the estrous cycle. Each value represents the mean ± SEM. N = 11 (P > 0.05)



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  		FIG. 2. GSH concentration of reproductive tract flushing collected at midday from Day 1 (oviduct), Day 2 (uterus), and Day 3 (uterus) of gestation following in vivo treatment with BSO (4 mmol/kg body weight/ injection) four times per day at 3-h intervals starting from Day 0 until collection of reproductive tract flushing. Each value represents the mean ± SEM. Letters indicate significant differences between days of gestation. Asterisks indicate significant difference between BSO and control within day. N = 11 (P < 0.05)

Embryo Development and Embryo GSH Concentrations after Treatment with BSO

To determine if GSH in RTS is important for embryo development in vivo under normal conditions, mice were treated with BSO during various stages of reproduction to block GSH synthesis and lower the concentration of GSH. When BSO was administered to mice after plug detection, there was no significant difference in embryo development on Days 1, 2, or 3 (Figs. 3–5), even though the concentration of GSH in the reproductive tract flushings was drastically decreased (Fig. 2). In addition, no significant differences were found in embryonic GSH concentration on Days 1 or 2 when BSO was administered after plug detection (Fig. 6). Day 3 is not included because embryos are synthesizing their own GSH by this stage and would not be relying on the reproductive tract for GSH.



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  		FIG. 3. Percent of embryos at various stages of development collected at midday of Day 1 of gestation after in vivo treatment with BSO (4 mmol/ kg body weight/injection) four times per day at 3-h intervals starting from Day 0 until collection of embryos. Each value represents the mean ± SEM. N = 12 (P > 0.05)



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  		FIG. 5. Percent of embryos at various stages of development collected at midday of Day 3 of gestation after in vivo treatment with BSO (4 mmol/ kg body weight/injection) four times per day at 3-h intervals starting from Day 0 until collection of embryos. Each value represents the mean ± SEM. N = 12 (P > 0.05)



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  		FIG. 6. GSH concentration in embryos collected at midday on Days 1 and 2 of gestation following in vivo treatment with BSO (4 mmol/kg body weight/injection) four times per day at 3-h intervals starting from Day 0 until collection of embryos. Each value represents the mean ± SEM. N = 7 (P > 0.05)

Development of Embryos Cultured in Reproductive Tract Flushings

To determine if decreasing the concentration of GSH in RTS has an effect on embryo development in vitro, embryos were cultured in medium supplemented with reproductive tract flushings or reproductive tract flushings from BSO-treated mice. Embryos were cultured in drops consisting of 50% M16 and 50% M16 reproductive tract flushing. There was no significant difference between the percent of embryos developing to the blastocyst stage in the control flushing group and the BSO reproductive tract flushing group (Fig. 7) even though the concentration of GSH in the flushing was decreased at least 75% (Fig. 2). However, there was a significant difference between the percent of embryos developing to the blastocyst stage in the control medium group compared with media supplemented with reproductive tract flushing (Fig. 7).



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  		FIG. 7. Percent of embryos developing to the blastocyst stage by the afternoon of Day 4 after being cultured for 3 days in reproductive tract flushings from mice treated with BSO (4 mmol/kg body weight/injection) four times per day at 3-h intervals starting from Day 0 until collection of reproductive tract flushing. Embryos were transferred to medium containing reproductive tract flushings corresponding to the day of development. Each value represents the mean ± SEM. Columns with different superscripts are significantly different. N = 15 (P <0.05)

BSO Administered Before Ovulation Decreases Development

These experiments were conducted to determine the effect inhibition of GSH synthesis before ovulation had on the concentration of GSH in the oocyte and further embryonic development in vivo (Figs. 8 and 9) Treatment of mice with BSO from 60 h before ovulation to injection of hCG decreased the GSH content of the oocyte and resulted in an increased percentage of degenerative embryos on Day 1 of development.



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  		FIG. 8. GSH concentration of oocytes from virgin females, 24 h after hCG injection after in vivo exposure to BSO (4 mmol/kg body weight/ injection) four times per day at 3-h intervals starting from 60 h before injection of hCG until the collection of oocyte. Each value represents the mean ± SEM. Asterisks indicate treatment is significantly different from control. N = 11 (P <0.05)



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  		FIG. 9. Percent of degenerate embryos collected at midday of Day 1 of gestation following in vivo treatment with BSO (4 mmol/kg body weight/ injection) four times per day at 3-h intervals starting from 60 h before injection of hCG until collection of embryos. Each value represents the mean ± SEM. Asterisks indicate treatment is significantly different from control. N = 9 (P <0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We investigated the possibility that GSH in RTS provides protection to the cleavage-stage embryo during its developmental period when it cannot synthesize GSH de novo [9]. Exposure of cleavage-stage embryos to GSH oxidizing and depleting agents in vitro decreases the intracellular GSH concentration and the development of the cleavage-stage embryo to the blastocyst stage [8, 9, 10]. Cleavage-stage embryos exposed to acetaminophen in vitro experience a decrease in intracellular GSH concentration as well as a decrease in development to the blastocyst stage; however, this acetaminophen-induced decrease in GSH concentration and development is absent when the cleavage-stage embryo is exposed to acetaminophen in vivo, suggesting some type of maternal protection mechanism [12]. Extracellular GSH has been shown to provide protection for other cell types and is found in RTS [13]; therefore, we hypothesized it may play a similar role in the protection of the cleavage-stage embryo.

Embryos allowed to develop in RTS with a decreased concentration of GSH, both in vivo and in vitro, did not exhibit a decrease in intracellular GSH concentration or development, leading to the conclusion that extracellular GSH found in the reproductive tract does not have a major role in development of the cleavage-stage embryo under normal conditions. It is possible that protection from endogenous ROS is provided to the embryo by other means. ROS are the product of endogenous processes and exogenous agents and act to increase oxidative stress within a cell, shifting the intracellular redox state to an oxidized status and increasing cellular injury [30, 31]. Superoxide dismutase, glutathione peroxidase, and catalase all act to reduce ROS and peroxides and exhibit increased activity within bovine oviducts over the phases of the estrous cycle [32]. These enzymes could provide protection to the embryo without GSH. Although glutathione peroxidase usually utilizes GSH as a reducing agent, in some circumstances where the concentration of GSH is low (such as in plasma), glutathione peroxidase can use thioredoxin and glutaredoxin instead of relying primarily on GSH [33]. Another GSH-associated enzyme, glutathione reductase (GR), is responsible for maintaining GSH levels by reducing GSSG back to GSH using NADPH. GR activity is higher in the oviduct than in the rest of the reproductive tract and could help provide protection to the cleavage-stage embryo by recycling GSSG back to GSH [34]. Another possibility could be that protection from ROS and toxicants is provided to the embryo by the cells lining the reproductive tract, preventing ROS and toxicants from reaching the embryo. Oviduct epithelial cells show an increase in GSH concentration during the luteal phase of the estrous cycle, a time when the embryo would be developing, and could provide a first line of defense for the embryo [35].

In vitro analysis of embryo development in reproductive tract flushings showed a decrease in development of embryos cultured in the presence of reproductive tract flushings, regardless of treatment, when compared with controls. This efffect could be due to a change in the concentration of nutrients in the culture media. M16 culture medium has been optimized for the support of embryo development in vitro. While RTS should be optimum for in vivo development, the conditions in vivo are not identical to the in vitro environment, so a different nutrient composition would be optimum. Adverse effects of uterine and oviduct flushings on preimplantation embryo development in vitro have been demonstrated in several other studies [36–40]. Another possible explanation for the decrease in embryo development in reproductive tract flushings in vitro is that RTS may contain nutrients or factors that are continually replenished in vivo but depleted in vitro due to the lack of the action by the cells of the reproductive tract. Preimplantation embryos cultured in the presence of oviductal cells show lower apoptosis and higher maintenance of mitochondrial function when compared with embryos cultured in only embryonic culture media [41, 42].

It is possible that the decrease in embryonic GSH concentration observed during preimplantation development or a shift to a more oxidized state of glutathione is important to normal differentiation and therefore there are mechanisms to inhibit uptake of GSH from RTS. Our data demonstrate a decrease in the concentration of GSH in RTS on Day 2 of gestation when the potential need for GSH is high because the embryonic GSH concentration is low and the embryos cannot synthesize GSH de novo. Perhaps a drop in embryonic GSH concentration on Day 2 of development is important for the differentiation and apoptosis of the cells within the blastocyst. An intracellular shift to a more oxidized status is associated with changes in enzyme activity, differentiation, and apoptosis in other tissues [43, 44].

It is possible that the amount of GSH in the reproductive tract even after treatment with BSO is sufficient to protect the preimplantation embryo under normal conditions and/ or the preimplantation embryo upregulates its own GSH protection mechanism to compensate for the decrease in reproductive tract secretion GSH. Preimplantation embryos exposed to tertiary-butyl hydroperoxide to cause oxidative stress, upregulated glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and developed the capacity to synthesize GSH de novo at the cleavage stage [45]. Therefore, the preimplantation embryo might rely on its ability to respond to oxidative stress by upregulation of GSH synthesis as opposed to relying on extracellular GSH within the reproductive tract. Some extracellular fluids, such as plasma, contain GSH concentrations that are considerably less, on a nmol GSH/mg protein basis, than that of BSO-treated RTS, but the cells within the plasma are able to synthesize GSH and they function normally [46, 47].

Although inhibition of GSH synthesis after fertilization did not affect the cleavage-stage embryo, a decrease in GSH concentration in the oocytes caused an inhibition in development after fertilization in vivo. Previous studies have found an inhibition in embryo development after depletion of oocyte GSH concentration followed by in vitro fertilization [3–5]. Our study shows similar results when oocytes were both depleted and fertilized in vivo and provides further support for the importance of maternal GSH stored in the oocyte for normal embryonic development.

In conclusion, examination of cleavage-stage embryo development in RTS with a decreased concentration of GSH showed that the embryo does not require most of the extracellular GSH found in RTS under normal conditions.



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  		FIG. 4. Percent of embryos at various stages of development collected at midday of Day 2 of gestation after in vivo treatment with BSO (4 mmol/ kg body weight/injection) four times per day at 3-h intervals starting from Day 0 until collection of embryos. Each value represents the mean ± SEM. N = 12 (P > 0.05)


   ACKNOWLEDGMENTS
 
The authors wish to thank the following people for their help and support: Sally Boyd, Christian Perry, Shawn Stover, and Michelle Palic.


   FOOTNOTES
 
1 Supported by the National Institute of Environmental Health Sciences, NIH grant ES08818. Back

2 Correspondence: Frank Skufca, University of Northern Colorado, Department of Biological Sciences, Greeley, CO 80639. FAX: 970 351 2335; frank.skufca{at}unco.edu Back

Received: 22 November 2004.

First decision: 14 December 2004.

Accepted: 29 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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