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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

April 2000
Volume IV, Number 2
ISSN: 1537-6583
Pages: 110-123

V. Zaken, R. Kohen and A. Ornoy

Laboratory of Teratology Dept. of Anatomy & Cell Biology, and Department of Pharmaceutics, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Short title: The antioxidant defense mechanism in rat embryos

Key words: SOD, CAT, LMWA, embryo, yolk sac, cyclic voltammetry

Acknowledgement: Supported by grant No 032-4781 from the Israeli Ministry of Health

Abstract

Reactive oxygen species (ROS) are involved in the etiology of numerous diseases and are suggested to be one of the mechanisms of action of several teratogens such as cocaine, high concentrations of glucose and ketone bodies. We studied the antioxidant capacity of 9.5-12.5 day old rat embryos and their yolk sacs both in vivo and in vitro. We measured the activity of superoxide dismutase (SOD) and the hydrogen peroxide removing activity (mainly due to catalase (CAT) and glutathione peroxidase (GSH -Px) and found significant activity of these enzymes already at day 9.5 in the embryos and their yolk sac, both in vivo and in vitro. A gradual increase in the activity was found with the advancement of embryonic age. The reducing power, that reflects the concentration of low molecular weight antioxidants (LMWA) was measured by cyclic voltammetry. LMWA were found in the embryos and their yolk sacs on days 9.5-11.5 of gestation with the peak potential of 0.56- 0.62 Volts. On day 12.5 an additional group of LMWA appeared at a peak potential of 0.95-0.97 Volts. There was a gradual increase in the concentration of LMWA with the increase in embryonic age. Generally, the concentration of LMWA was higher in the embryo than in its yolk sac but it was similar in vivo and in vitro at the same developmental stage. The gradual development of the embryonic antioxidant capacity implies that under normal conditions the developing embryo is capable of coping with oxidative stress, but this may fail under various pathological conditions, leading to embryonic damage.

Introduction

Reactive oxygen species (ROS) are involved in the etiology of numerous diseases such as artheriosclerotic cardio-vascular diseases, ischemic diseases and aging processes (Gutteridge 1993; Halliwel et al., 1992; Halliwel and Gutteridge 1995). ROS are involved in the etiology of diabetes mellitus and are produced in large amounts in various metabolic disorders (Papaccio et al., 1986). It has recently been suggested that ROS have an important role in the etiology of several congenital anomalies such as those produced by cocaine (Fantel et al., 1992; Zimmerman et al., 1994), and maternal diabetes (Eriksson and Borg 1993; Ornoy et al., 1996; Wentzel et al., 1997). ROS have also been implicated in the etiology of cellular damage induced by ionizing irradiation (Anand et al., 1997). The same mechanism may be involved in radiation induced teratogenesis.

The antioxidant defense mechanisms include several enzymes, the most important of them are superoxide dismutase (SOD) that produces hydrogen-peroxide from superoxide radicals, catalase (CAT) and glutathione peroxidase (GSH-Px) which decompose hydrogen-peroxide (Chevion 1988; Halliwell 1990; Hass et al., 1989; Trocino et al., 1995). In addition, there are low molecular weight antioxidants (LMWA) which act directly with various ROS, such as Vitamin C, Uric Acid, glutathione and vitamin E (Sharma and Buetner 1993). These are ROS scavengers.

The antioxidant defense mechanism can be studied by numerous methods. While there are well established methods for quantification of the antioxidant enzymes, methods for the evaluation of the total LMWA are scarce. Recently, a new method based on the cyclic voltammery (CV) for evaluation of the overall antioxidant activity of the LMWA was developed in our laboratory (Chevion et al., 1997; Guadalupe 1992; Kohen 1992; 1993; Kohen et al. 1997). The principle of the method is based on the fact that LMWA which act directly on ROS are reducing equivalents capable of donating their electrons to the ROS and neutralizing them. Therefore, measurement of the overall reducing power of a biological tissue or fluid would reflect its antioxidant activity. This method has been demonstrated to reflect the overall LMWA activity of biological tissues and fluids in various systems.

The CV by itself cannot provide specific information on the exact nature of the LMWA. It can, however, supply information concerning the type of various antioxidants in the sample and their total concentration (see methods).

The developing embryo uses during early development both aerobic and anaerobic metabolic pathways. ROS may therefore be produced at early developmental stages, necessitating anti-oxidative defense mechanisms (Allen and Balin 1989; El Hage and Singh 1990; Frank 1991; Ornoy et al., 1996; Parchment 1992). The question related to the nature of these antioxidant defense mechanisms and the developmental stage at which they first appear is therefore of significant importance. We have studied the presence of LMWA in 2-4 cell stage mouse embryos and in blastocysts both in vivo and in vitro using cyclic voltammetry (Ornoy et al., 1996).

We found that even the 2 cell stage pre implantation embryo has a significant reducing power; the peak potential (i.e. biological oxidation potential) measured as half of the anodic waves ranges between 0.7-0.8 V, and the concentration of LMWA (as measured by the anodic current) was found to be about 2.0 m A.

Watson et al.(1997) studied the expression of SOD in human villous trophoblastic cells in relation to gestational age from the 8^th week of pregnancy to term. They found by immunostaining expression of SOD in the cytotrophoblastic cells as well as in the syncytiotrophoblastic cells. In the latter cells the immunostaining for SOD increased until week 14 of gestation (Watson et al 1997).

The purpose of the present study was to look at these parameters during 9.5-12.5 days of gestation in rat embryos immediately after their removal from the uterus (in vivo) and in 10.5 days old embryos cultured for 24 or 48 hours (in vitro).

Material and Methods

We studied 9.5 -12.5 day old rat embryos and their yolk sacs, which were removed from the uterus. The day sperm were found in the vagina was considered to be day 0. Embryos of 9.5 and 10.5 days were studied with their yolk sacs. Embryos of 11.5 and 12.5 days were separated from their yolk sacs with the embryos and yolk sacs being studied separately.

10.5 days old rat embryos were also removed for whole embryo culture. They were prepared for culture according to the method described by New (1978) and cultured in normal rat serum for 24 or 48 hours. Each embryo was cultured in 0.9ml rat serum and 0.1 ml distilled water (Zusman and Ornoy 1987). Following culture, the embryos were scored according to the method described by Brown and Fabro (1981) and screened for anomalies. Only normal embryos with a beating heart and intact yolk sac circulation were used for further studies. The mean morphologic score of 10.5 days old embryos cultured for 24 hours was 39.5 ± 0.2 (range: 36.5-42.5), for 48 hours it was 60.2± 0.2 (range 51.0-61.0).. Embryos and yolk sacs at various gestational days were homogenized (each embryo and/or yolk sac in 1 ml PBS) using a Vortex Homogenizer and stored in -70° C, until further study.

The antioxidant defense mechanism of each embryo and/or yolk sac was studied by examining SOD activity, hydrogen peroxide removing activity (which is mainly as a result of CAT and GSH Px activity) and by cyclic voltammetry for the evaluation of the low molecular weight antioxidants. Results were expressed per embryo and/or yolk sac and per µ g protein. Enzyme activity was studied for 3 minutes and was expressed in Units ( activity per minute).

Protein determination: This was performed according to the method described by Bradford; i.e. spectrophotometric determination of the complex of protein and the reagent (dye with phosphoric acid and methanol) (Bradford 1976).

Cyclic Voltammetry: The reducing power of the embryos and/or yolk sacs (both in vivo and in vitro) was studied on tissue homogenates. In order to evaluate the total reducing power we have used a cyclic voltammeter (BAS, West Lafayette, IN) model CV-1B. All cyclic voltammograms were performed between - 200 mV and 2.0 V.

Figure 1 shows a typical cyclic voltammogram (CV) of a 12.5 days old embryo homogenate. As shown in the CV the peak potential is calculated from the potential applied on the working electrode (X-axis). This potential which is calculated at half the increase of current at the anodic wave is typical of the tested embryo and represents the ability of the reducing equivalents composing the wave, to donate their electrons to the working electrode. The wave recorded may contain one or several reducing LMWA possessing a specific peak potential. If another group of LMWA is also present, they may appear as another wave at a different potential. The lower the potential, the higher the ability of the compounds responsible for the wave to donate their electrons, indicating stronger reducing power. The buffer itself did not possesses any reducing properties. Another parameter which can be calculated from the CV is the total concentration of the reducing LMWA composing the anodic wave. This can be achieved by calculating the anodic current from the y-axis. The current produced in the wave is proportional to the concentration of LMWA. A decrease in the anodic current may indicate a reduction in the levels of the LMWA, while an increase indicates an increase in their concentration. Appearance of new anodic waves indicate the induction of a different type of LMWA.

The peak potential (the capacity of LMWA to transfer electrons) was measured in volts on the X-axis and the concentration of LMWA was measured in µ A on the Y axis (figs 1,2). The measurements were carried out at 37º C in 0,01M phosphate buffered saline, pH 7.4. We used three electrodes for this study. The working electrode was a glassy carbon disk 3.3 mm in diameter (BAS MF - 2012, Bioanalytical systems Inc.). The auxiliary electrode was a platinum wire, and the reference electrode was Ag/Ag C1 (BAS). The working electrode was polished before each measurement with polishing kit (BAS - Bioanalytical systems, Inc.). The method was described in details by Kohen and by others (Kohen 1992; 1993; 1997; Chevion et al. 1997; Gliadalupe 1992).

Detection of antioxidant enzyme activities
SOD activity was studied by the method described by McCord and Friedovich and modified by Grankvist et al. (1981; 1988). Xantine oxidase added to the solution produces ROS. Cytochrome C was used as a detector. The superoxide radicals reduce the cytochrome C, and the reduced cytochrome C is measured by spectrophotometry at 550nm. The addition of SOD will reduce the amount of superoxide radicals, thus reducing the amount of reduced cytochrome C which is detected by the spectrophotometer. SOD activity was expressed in Units, calculated from the slope obtained when OD was plotted Vs time.

Hydrogen peroxide removing activity: The method we used to detect the hydrogen peroxide removing activity of both enzymes was described by Thurman et al.(1972). Aliquots of 20 m l of the homogenate were added to the reaction mixture containing hydrogen peroxide at a concentration of 1 mM. Following 10 minutes of incubation the reaction was stopped by addition of 200 m l TCA (30%) and the remaining hydrogen peroxide was determined according to the Thurman procedure (1972). In brief, it measures the red complex that is formed by hydrogen peroxide, ferrous amonium sulfate and thiocyanate. The concentration of the complex is read by a spectrophotometer at 480 nm, which is in direct relation to the concentration of hydrogen peroxide in the tested solution. A calibration curve of catalase was prepared separately and the results were calculated as “catalase like” activity. Hydrogen peroxide removing activity, mainly induced by CAT and GSH- Px reduce the amount of hydrogen peroxide, reducing the concentration of the complex.

Statistical analysis: This was performed by using Students t test (two tailed) and Chi square. Significance was set at P= 0.05

Results

The results of the C V are described in Tables 1 and 2 and in Figures 1 and 2. In the embryos in vivo obtained with their yolk sacs from pregnant rats at days 9½ and 10 ½ we found that the peak potential was similar in these 2 days, being 0.56-0.59V (Table 1). A similar but slightly higher oxidation potential was found in the embryos and yolk sacs (separately) on day 11½ of gestation (Table 1), being 0.61-0.62V. The anodic current increased with the advancement of embryonic age, in both the embryos and the yolk sacs, implying a gradual increase in the concentration of LMWA in these embryos. The anodic current was higher in the embryo when compared to the yolk sac of the same age (Table 1). In the embryos and their yolk sacs on day 12½ of gestation a second anodic wave appeared on the X axis at 0.95-0.98 V, implying that at that age there is an additional group of LMWA in the embryo. The anodic current corresponding to that second wave was of the same magnitude as that of the first wave (Table 1, Figure 1).

Table 2 and Figure 2 show the results of the CV of rat embryos cultured in vitro for 24 hours (compared to 11½ days old embryos in vivo), and for 48 hours (compared to 12½ days old embryos in vivo). The peak potential of the embryos and yolk sacs after 24 hours of culture was slightly higher than at that age in vivo, but the differences were not statistically significant. After 48 hours of culture, two waves appeared on the X axis, similar to those found in 12½ days old embryos in vivo (Tables 1,2).

The anodic current in embryos and yolk sacs after 24 hours of culture was similar to 11½ days old embryos in vivo. After 48 hours in culture the anodic current corresponding to the first wave in the embryo was lower than that obtained in vivo, but the anodic current of the second wave was similar.

The SOD activity in the embryos and their yolk sacs in vivo is given in Table 3. There is an increase in the inhibition of cytochrome C reduction with advancement of embryonic age in the embryos and their yolk sacs, implying increased SOD activity. This increased activity is observed when expressed in units of SOD per mg protein and as SOD units per embryo and/or yolk sac (Table 3). Table 4 shows the results of SOD activity in 10½ day old rat embryos and yolk sacs cultured in vitro for 24 and 48 hours. The results are similar to those obtained in 11½ and 12½ days old rat embryos in vivo (Tables 3,4).

The hydrogen peroxide removal activity in vivo and in vitro are described in Tables 5 and 6, which give the hydrogen peroxide removed per embryo and/or yolk sac in each of the studied days. In vivo there is a significant increase in the amount of hydrogen peroxide removed with the increase in embryonic age, mainly implying a higher activity of CAT and GSH- Px with advancement of embryonic age (Table 5). The increase in activity is demonstrated also when the results are described per mg protein. Similar results are obtained in 10½ day old rat embryos following 24 and 48 hours of culture (Table 6). Hydrogen peroxide removal activity after 24 hours of culture is similar to that on 11½ days in vivo and after 48 hours is similar to that observed in 12.5 days old embryos and yolk sacs in vivo.

Discussion

We studied the antioxidant capacity of 9 .5-12 .5 day rat embryos and their yolk sacs in vivo and in vitro. A gradual increase in the activity of SOD and of hydrogen peroxide removal activity (mainly produced by CAT and GSH-Px) was observed with the increase in embryonic age. The activity of SOD and the hydrogen peroxide removal activity were similar in vivo and in vitro. When calculated per milligram protein the activity of SOD was similar in the embryos and in the yolk sacs, while the activity of other peroxidases was higher in the embryo in comparison to the yolk sac. Low molecular weight antioxidants were found in the embryos and their yolk sacs already at 9 .5 days of gestation. At 12 .5 days, an additional group of LMWA appeared, as observed by cyclic voltammetry, possessing a peak potential of 0.95-0.98 V.

There is a changing need for oxygen in the developing embryo at different ages. In the blastocyst, relatively high oxygen tension is toxic. However, when embryonic and fetal circulation are established, the embryo is exposed normally to higher concentrations of oxygen (Alen and Balin 1989; Miki et al., 1988). During that time, the embryo must develop its antioxidant defense mechanism, slowly increasing the activity of the major antioxidant enzymes (Wentzel et al., 1997). Indeed, mRNA for Cu/Zn SOD was observed in 8 day old mice embryos and was found to increase with the increase in embryonic age (El Hage and Singh 1990). We also found a gradual increase in the activity of these antioxidant enzymes in relation to embryonic age in vivo and in vitro.

Very high levels of oxygen and other ROS are toxic to the embryo and fetus, apparently due to the fact that the superoxide radicals created in such a condition are in excess in relation to the antioxidant capacity of the developing embryos. This may lead to the production of highly reactive oxygen species, inducing oxidative stress and embryonic damage (Miki et al., 1988; Fantel et al., 1992).

Too low oxygen concentrations may also be embryotoxic, as the developing embryo needs optimal oxygen concentrations to meet his energy needs (Miki et al., 1988).

SOD activity was found to be cell cycle dependent decreasing during mitosis, while GSH synthesis was found to increase during mitosis (Allen 1991). As the activity of antioxidant enzymes is influenced by ROS production, it is presumed that ROS have an important role in the process of embryonic development by altering gene expression during development as they may affect the cytoskeleton, nuclear matrix proteins, chromatin configuration and cellular ion distribution (Allen 1991). A substantial change in ROS production may therefore have a significant impact on embryonic development.

LMWA are a group of compounds designed to cope directly with the high levels of ROS. This group contains a large number of molecules derived from various sources. These include substances that can be produced by the living cell itself (such as GSH), waste products of the living cell which possess antioxidant activity such as uric acid and a majority of compounds originating from the diet i.e. vitamin C and vitamin E (Halliwel and Gutteridge 1995; Sharma and Buetner 1993).

The reducing power which reflects the concentrations of LMWA was measured by CV. In previous studies we have found that LMWA can be observed even in 2-4 cell stage mouse embryos (Ornoy et al., 1996). The reducing substances found in 9 .5 day old rat embryos and yolk sacs showed a peak potential of about 0.6 volts, while in blastocysts it was higher, about 0.8 volts (Ornoy et al., 1996). This change may be due to the presence of a different group of LMWA in the blastocysts or a significant change in the relative concentrations of the LMWA composing the two waves. Such a change may alter the peak potential. Although the nature of these reducing compounds is not known yet, they probably belong to the LMWA which are synthesized by the cell itself. The observed peak potential may suggest that the compound composing the wave may be ascorbic acid, uric acid NADH, NADPH, or other related compounds. The anodic current was also different in blastocysts as opposed to 2 cell stage embryos but the main difference was in comparison to elder, 12 .5 days old embryos. In the present study we found that the anodic current increased with age in both the embryos and their yolk sacs, reaching 4 m A at 12.5 days. This implies an increase in the concentration of LMWA in the embryo and yolk sac with the increase in gestational age, enabling the conceptus to cope better with oxidative stress. A similar embryonic protective mechanism was observed by Barnea et al. (1993,1995). They found quinone reductase activity in first trimester human placenta as well as in porcine liver embryonic cells. Quinone reductase is known to transform unstable quinones into relatively stable hydroquinones, thus protecting the developing embryo from toxic environmental agents (Barnea et el, 1993.1995).

The fact that at 12 .5 days of gestation there are 2 waves in the embryos and in the yolk sacs implies that at that stage of development there are at least 2 groups of reducing substances which serve as LMWA. Preliminary studies using HPLC have shown that some of these LMWA composing the first anodic wave are ascorbic acid and uric acid (Zaken et al,1999). Moreover, when we cultured 10.5 day old rat embryos in diabetic serum we found a decrease in LMWA, in SOD and CAT activity in the embryos and yolk sacs with about 60% of the embryos exhibiting various congenital anomalies. When we added vitamin C and vitamin E to the diabetic culture medium all pathological changes in the embryos dissapeared (Zaken et al,1999). This ephasizes the important role of the antioxidant defense mechanism in the pathogenesis of various teratogens and the possibility to prevent some of these anomalies by modifying the antioxidant defense mechanism in the embryo.

It is suggested that the activity of SOD increases during embryonic development because it is stimulated by an increase in ROS generation (Allen 1991). An increase in ROS generation will probably affect the entire antioxidant defense mechanism. It is therefore reasonable to assume that the concentrations of the LMWA as well as the activity of the antioxidant enzymes will further increase with the advancement of embryonic age. It is also expected that with the increase in age, activity of the antioxidant enzymes will be higher in specific embryonic organs such as liver, lungs, heart and muscle than in other organs. It seems therefore that any pathological increase in the production of ROS (such as produced by increased oxygen or by several teratogens: i.e.diabetes) may, in response, increase the LMWA concentrations and the activity of antioxidative enzymes. If ROS production is too high, that increase will not be sufficient to meet the needs and the developing embryo. This may deplete the LMWA and reduce the activity of antioxidant enzymes as indeed found by us in diabetes. As a result the embryo may be damaged resulting in congenital anomalies or death. Further studies are needed to elaborate these issues.

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Figure 1

Cyclic voltammogram of a 12.5 day old embryo removed from the uterus, homogenized and studied for its peak potential (biological oxidation potential). Two waves are observed on the X axis, corresponding to 2 peak potentials at 0.57 V and at 0.98 V implying that there are 2 groups of LMWA. The anodic current of these 2 peak potentials (Y axis) is similar, about 4 µA (arrows).

Figure 2

Cyclic voltammogram of a 10.5 day old embryo cultured in vitro for 48 h, corresponding to a 12.5 day old embryo in vivo, showing similar peak potentials as in figure 1. The first peak potential is at 0.45 V and the second at 0.80 V. The anodic current in this embryo also varies between the two peaks, being 2.9 µA in the first wave and 4,4 µA in the second.

Average Reducing power as reflected by the anodic current and peak potential ¹ in embryo or yolk sac cultured for 24h or 48h

Activity of SOD in the embryo and yolk sac in vivo according to embryonic age (Cytochrome C Method).

* Mean ± SE
^a Significantly higher than 11.5 days sac p < 0.05 (by chi square test).
^b Significantly higher than 11.5 days embryo. p < 0.05 (by chi square test).

Table 4

Activity of SOD in 10.5 days embryo and yolk sac cultured for 24h or 48h

Table 5

Hydrogen peroxide removing capacity of the embryo and yolk sac according to embryonic age in vivo.