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Endocrinology, doi:10.1210/en.2008-0187
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Endocrinology Vol. 149, No. 9 4428-4434
Copyright © 2008 by The Endocrine Society

Regulation of Key Antioxidant Enzymatic Systems in the Sheep Endometrium by Ovarian Steroids

Kaïs H. Al-Gubory, Philippe Bolifraud and Catherine Garrel

Unité de Biologie du Développement et de la Reproduction (K.H.A., P.B.), Département de Physiologie Animale, Institut National de la Recherche Agronomique (INRA), 78352 Jouy-en-Josas cedex, France; and Laboratoire de Biologie du Stress Oxydant (C.G.), Département de Biologie Intégrée, Centre Hospitalier Universitaire de Grenoble, 38043 Grenoble cedex 9, France

Address all correspondence and requests for reprints to: Kaïs H. Al-Gubory, Unité Mixte de Recherche Biologie du Développement et de la Reproduction, Institut National de la Recherche Agronomique (INRA), 78352 Jouy-en-Josas cedex, France. E-mail: kais.algubory{at}jouy.inra.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) and their control by antioxidant enzymes are involved in the physiology of the female reproductive system. Thus, it is important to understand the regulation of key antioxidant enzymatic pathways. The roles of estrogen and progesterone in regulating the physiological functions of the endometrium have become central dogma. We examined the effects of ovarian steroids on superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) activities in the aglandular caruncular and glandular inter-caruncular endometrial tissues of ovariectomized (OVX) ewes and in OVX ewes treated with estradiol (E2), progesterone (P4), or both hormones according to schedules designed to produce physiological changes of these hormones during the estrous cycle. The activities SOD2, CAT, GPX and GSR in both endometrial tissues were unaffected by P4 treatment. The activity of SOD1 in the aglandular tissue was unaffected by P4 treatment, however this treatment decreased SOD1 activity in the glandular tissue (P < 0.01). Treatment with E2, either alone or in combination with P4, decreased SOD1 (P < 0.01), CAT (P < 0.01) and GPX (P < 0.05) activities in both endometrial tissues. The activity of GSR decreased only in the glandular tissue (P < 0.05) after E2 treatment, either alone or in combination with P4. No change in SOD2 activity was detected in both endometrial tissues after administration of E2, P4 or both hormones. This study provides the first firm evidence for the role of ovarian steroid hormones in the regulation of the activities of key antioxidant enzyme in the endometrium of female mammals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REACTIVE OXYGEN SPECIES (ROS) are formed as by-products of aerobic metabolism, and mammalian cells have evolved a variety of enzymatic mechanisms to control ROS production (1). Copper, zinc-superoxide dismutase (SOD1), and manganese-SOD (SOD2) contribute to the first line of antioxidant pathway by catalyzing the conversion of superoxide radical (O2·–), into hydrogen peroxide (H2O2). Glutathione peroxidase (GPX) and catalase (CAT) both belong to the secondary antioxidant pathway by catalyzing the conversion of H2O2 to H2O. Glutathione reductase (GSR) catalyzes glutathione disulfide to reduced glutathione (GSH), which is an important component of antioxidant enzymatic systems. Indeed, GPX reduces H2O2 or other hydroperoxides using GSH as donor substrate. It follows that SOD1, SOD2, CAT, GPX, and GSR represent coordinately operating antioxidant systems to ensure efficient control of ROS production and cell signaling (2, 3).

There is convincing evidence that ROS affect physiological processes such as oocyte maturation, ovulation, follicular and luteal steroidogenesis, implantation, and early embryo development (4, 5). Although there is evidence for the involvement of ROS-scavenging enzymes in the uterine physiology, the role of ROS and antioxidant enzymes in endometrial function has not been fully understood. In the rodent uterus, superoxide radical and SOD levels exhibits cyclic changes during the estrous cycle, suggesting that superoxide may be involved in regulating cell proliferation of the uterus (6). A possible role of ROS and their scavenging systems in endometrial function was suggested by data obtained in human showing positive immunostaining for SOD1 and SOD2 in endometrial epithelium throughout the entire menstrual cycle (7). Total SOD activity in the endometrium increased from early proliferative phase to mid-late proliferative phase and further increased in the mid-secretory phase, then decreased in the late secretory phase. Furthermore, lipid peroxide is generated in the human endometrium and increased in the late secretory phase just before menstruation (7). It is therefore suggested that the decrease in SOD activity and the increase in lipid peroxide may contribute to the shedding of the human endometrium. These studies support the hypothesis that the first line of antioxidant system serves important physiological roles within the endometrium. GSR is also known to be present in the female rodent reproductive system, and marked changes in GSR activity were observed during the estrous cycle (8).

The uterus response to the varying levels of ovarian steroids is exhibited as changes in endometrium morphology and function during the luteal phase of the estrous (9) and menstrual (10) cycle. Although there is evidence to support regulation of SOD in murine uterine tissues by estrogen (11) and in human endometrial stromal cells by medroxyprogesterone acetate (12), no information has been available so far regarding the physiological in vivo regulation of key antioxidant enzymes in the mammalian female genital tract, particularly the involvement of ovarian steroid hormones. Study of this subject is particularly important because ROS and their scavenging systems may have a regulatory role in the uterine physiology (6, 7). Given the distinct morphology and function of aglandular and glandular endometrial tissues in sheep (13, 14), these areas were used in the present study to determine the individual or combined effects of E2 and P4 administrated to ovariectomized (OVX) ewes on the activities of key ROS-scavenging antioxidant enzymes, SOD1, SOD2, CAT, GPX, and GSR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and surgery
All experiments and surgical procedures were in accordance with the guide for the care and use of animals and were approved by the French Ministry of Agriculture according to the French regulation for animal experimentation (authorization no. 78-34). Sixteen ewes of the Préalpes-du-Sud breed were OVX and allowed to recover for 42 d before steroid treatments were begun. On the day of surgery, ewes were initially anesthetized with a mixture of pentobarbital (Sanofi, Paris, France) and thiopentone (Abbott, Aubervilliers, France). After endotracheal intubation, general anesthesia was maintained by constant inhalation of a mixture of oxygen and halothane. Reproductive organs were exposed through a midventral laparotomy, and the ovaries were removed. All ewes were injected with penicillin (106 IU/d) for a consecutive 3 d after surgery. Throughout the experiment, the ewes were housed in a well-ventilated building under conditions of natural day-length and temperature.

Steroid treatment
At 42 d after ovariectomy, all ewes were pretreated with E2 for 2 d (3 x 16 µg/d) to produce ovulatory E2 surge. Ewes were weighed (55 + 1.3 kg) and allocated at random to four groups (n = 4 ewes per group): control ewes (C) injected with 90% corn oil:10% ethyl alcohol, E2-treated, P4-treated, and E2/PR-treated ewes. The schedule of steroid hormone dose and days of injection is shown in Table 1Go. This steroid hormone administration protocol has been shown to produce physiological blood concentrations of E2 and P4 (15) corresponding to those during the follicular and luteal phases in intact cyclic ewes (16). All steroid hormones treatment were administrated in 1 ml of 90% corn oil:10% ethyl alcohol at intervals of 8 h by im injection.


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  		TABLE 1. P4 and E2 treatment regimen

 
Blood and tissue collection
Blood samples were taken from the jugular vein into evacuated heparinized tubes. After centrifugation (3000 x g, 4 C) for 30 min, plasma was stored at –20 C until assayed. After 12 d of E2, P4, or E2/P4 treatments, the ewes were killed at a local abattoir in accordance with protocols approved by the local institutional animal use committee. The reproductive tracts were collected and immediately transported to the laboratory. Caruncular and intercaruncular endometrium tissues were dissected separately, immediately snap-frozen in liquid nitrogen, and then stored at –80 C until processed for enzyme activities. For morphological analysis, sample of caruncular and intercaruncular zones were fixed overnight in 4% paraformaldehyde in PBS (PBS), pH 7.4, washed in PBS, dehydrated through a series of increasing concentrations of ethanol (70–100%), cleared in toluene, and embedded in paraffin wax. Sections of 6 µm were obtained, processed for routine histology, and stained with hematoxylin and eosin.

Antioxidant enzyme activity assays
Aglandular caruncular and glandular intercaruncular endometrium tissues were homogenized separately in cold phosphate buffer (50 mM, pH 7.4) and then the homogenates were centrifuged at 15,000 x g for 30 min at 4 C. The resulting supernatant was used for determination of protein concentrations and measurement of enzymatic activities. Protein concentrations were determined by Lowry’s method (17). Enzyme activities of SOD1, SOD2, CAT, GPX, and GSR in the supernatant of both endometrium tissues were determined as described previously in detail (18). Total SOD activity was measured using the pyrogallol assay based on the competition between pyrogallol oxidation by superoxide radicals and superoxide dismutation by SOD. To measure SOD2 activity, sodium cyanide was included to inhibit SOD1 activity. SOD1 activity was calculated by subtracting SOD2 activity from total SOD activity. The rate of auto-oxidation is taken from the increase in the absorbance at 420 nm. One unit of SOD activity is defined as the amount of the enzyme required to inhibit the rate of pyrogallol auto-oxidation by 50%. CAT activity was measured by a simple and rapid method. The rate of hydrogen peroxide decomposition by CAT was followed at 240 nm. One unit was defined as the decomposition of 1 mmol hydrogen peroxide/min/mg protein. GPX activity was measured using the glutathione reductase-reduced nicotinamide adenine dinucleotide phosphate (NADPH) method. Enzyme activity was determined by a coupled assay system in which oxidation of glutathione was coupled to NADPH oxidation catalyzed by glutathione reductase. The rate of glutathione oxidized by tertiary butyl hydroperoxide was evaluated by the decrease of NADPH in the presence of EDTA, excess reduced glutathione, and glutathione reductase. The rate of decrease in concentration of NADPH was recorded at 340 nm. GPX activity was expressed in terms of nanomolar concentration of NADPH oxidized per minute per milligram of protein. GSR activity was assayed by the standard method of NADPH oxidation. In this assay, oxidized glutathione is reduced to glutathione by GSR, which oxidizes NADPH to NADP+. NADPH consumption was determined at 340 nm. Enzyme activity was expressed in terms of nanomolar concentration of NADPH oxidized per minute per milligram of protein.

Steroid hormone assays
Plasma samples were analyzed for P4 concentration in duplicate by a RIA as previously described (19) and validated for sheep jugular venous plasma with slight modifications (20). Briefly, dextran-coated charcoal solution was used instead of polyethylene glycol for the separation of bound and free radioactivity. Tritiated P4 (1,2,6,7–3H-progesterone, sp act 88 Ci/mmol) was obtained from Amersham, and a specific anti-P4 antibody was obtained from the Institut Pasteur (Paris, France). Hormone preparations, P4 tracer, and other reagents were diluted in 0.1 M PBS (pH 7.25). Tritiated P4 (3000 cpm) in 100 µl buffer, 100 µl P4 antiserum (1/20 000 dilution), 20 µl P4 standard or plasma samples, and 80 µl buffer were dispensed into the assay tubes. After an initial 2-h incubation at laboratory temperature (21 C) followed by 24-h incubation at 4 C, 2. 2 ml cold charcoal-dextran solution containing 0. 1% gelatin was added, and the tubes were centrifuged at 3000 x g for 45 min. The supernatant was decanted into vials, and 3 ml scintillation fluid (Scintillator Plus; PerkinElmer Life & Analytical Sciences, Boston, MA) were added to each vial. The radioactivity was counted in a Packard Tri-Carb Liquid Scintillation analyzer (model 2100 TR; Groningen, The Netherlands). To minimize assay variability, all plasma samples were analyzed in a single RIA. The limit of assay sensitivity was 0.1 ng/ml, and the intraassay coefficient of variation was less than 10%.

E2 was measured by RIA using an antiserum raised in rabbits against a conjugate of 17β-estradiol-6-O-carboxymethoxime with BSA (21). Tritiated estradiol ([1,2,6,7–3H]-estradiol, sp act 96 Ci/mmol) was obtained from Amersham Biosciences (Amersham, Buckinghamshire, UK). This RIA was a simple ether extraction of human serum as previously described (22) and validated for sheep serum (23) except for minor differences in the extraction procedure. Hormone preparations, E2 tracer and other reagents were diluted in 0.05 M PBS (pH 7). Plasma (1 ml) was extracted twice with 4 vol of purified diethyl ether. The samples were vortexed for 4 min and then the two phases were allowed to separate. The aqueous phase was frozen at –20 C and the ether extract was decanted into glass tubes. The organic phase was evaporated to dryness under nitrogen and the dried extracts were dissolved in 0.3 ml PBS containing 0.1% (wt/vol) gelatin and then assayed for E2. Approximately 2000 counts per minute tritiated E2 in 100 µl buffer, 100 µl E2 antiserum (1/80,000 dilution), 20 µl E2 standard or ether-extracted plasma samples, and 80 µl buffer were dispensed into the assay tubes. After 1-h incubation at 37 C, the tubes were placed on crushed ice to stop the reaction. Then 2.4-ml scintillation fluid (Lipoluma Plus; PerkinElmer Life & Analytical Sciences, Boston, MA) were added to each tube. The aqueous phase was frozen at –20 C and the scintillate phase (unbound radioactivity) was decanted into vials. The radioactivity was then counted in a Packard Tri-Carb Liquid Scintillation analyzer. All plasma samples were run in duplicate in a single RIA. The minimum detectable concentration of E2 was 0.2 pg/ml, and the intraassay coefficient of variation was less than 10%.

Statistical analysis
Data were analyzed by ANOVA and the new Duncan’s multiple range test (PRISM Graph Pad version 2; Graph Pad Software, San Diego, CA). The acceptable level of significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The concentrations of E2 (pg/ml) and P4 (ng/ml) in the jugular venous plasma of the control OVX ewes, and OVX ewes treated with E2, P4, or both steroid hormones are shown in Fig. 1Go, A–D. In all the OVX ewes, the administration for 2 d of 16 µg E2/d produced a preovulatory surge of 27.9 ± 4.5 pg/ml (mean ± SEM, n = 16 ewes), a level relatively similar to that reported in intact ewes (15). After this surge, the E2 and P4 administration protocol (Table 1Go) produced an episodic-like pattern of peripheral hormone concentrations. In OVX ewes treated with E2 alone (Fig. 1BGo) or in combination with P4 (Fig. 1DGo), plasma concentration of E2 showed a cyclic pattern and three peaks of E2 were detected on d 2–4, 6–8, and 10–12 after treatment. In OVX ewes treated with P4 alone (Fig. 1CGo) or in combination with E2 (Fig. 1DGo), plasma concentrations of P4 steadily increased from d 1 to d 12 after treatment. Overall, the pattern of changes in the plasma concentrations of E2 and P4 were almost identical to those observed during the follicular and luteal phases of the estrous cycle in intact ewes (15).


Figure 1
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  		FIG. 1. Plasma concentration of P4 ({circ}) and E2 (bullet) in control OVX ewes treated with 90% corn oil:10% ethyl alcohol (Fig. 1AGo), and in OVX ewes treated with E2 (Fig. 1BGo), P4 (Fig. 1CGo), or both hormones (Fig. 1DGo). At 42 d after ovariectomy, all ewes were pretreated with E2 for 2 d (3 x 16 µg/d) to produce ovulatory E2 surge. Ewes were then allocated at random to four groups (n = 4 ewes per group). The schedule of steroid hormone dose and days of injection is shown in Table 1Go. All steroid hormones treatment were administrated in 1 ml of 90% corn oil:10% ethyl alcohol at intervals of 8 h by im injection. Values are means for the number of animals used per group.

 
Although caruncular and intercaruncular areas of the sheep endometrium are visible to the naked eye (Fig. 2AGo), we checked using routine histology the accuracy of the dissection to separate the two morphologically different endometrial areas. Light microscopic examination showed that the caruncular area has luminal epithelium, compact stroma and was devoid of uterine glands (Fig. 2BGo), whereas the intercaruncular area contains large numbers of uterine glands (Fig. 2CGo).


Figure 2
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  		FIG. 2. Endometrium surface of ovariectomized ewe showing irregular caruncular zones (car) separated by intercaruncular zones (icar) (Fig. 1AGo). Representative photomicrographs illustrating general histo-architecture of the aglandular caruncular zones (Fig. 2BGo) and glandular intercaruncular zones (Fig. 2CGo) from an ovariectomized ewe. Note that the caruncular area has luminal epithelium (e), compact stroma (s) and devoid of uterine glands (g), whereas the intercaruncular area contains large numbers of uterine glands. Sections (6 µm) were prepared and stained with hematoxylin and eosin. Magnification, x250.

 
The activities of SOD1, SOD2, CAT, GPX, and GSR were not significantly different between the aglandular caruncular and glandular intercaruncular endometrial tissues collected from control OVX ewes. The activities of SOD1, SOD2, CAT, GPX, and GSR in aglandular tissue were unaffected by treatment of OVX ewes with P4, as compared with those in control OVX ewes (Fig. 3Go, A–E). The activities of SOD1, CAT, and GPX significantly decreased in aglandular tissue of OVX ewes that received E2, either alone or in combination with P4, as compared with those in control OVX ewes (Fig. 3Go, A, C, and D). No significant change in the activities of SOD2 and GSR was detected in aglandular tissues after treatment of OVX with E2 alone or in combination with P4 (Fig. 3Go, B and E).


Figure 3
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  		FIG. 3. Activities of antioxidant enzymes (A, SOD1; B, SOD2; C, CAT; D, GPX and E, GSR) in the sheep aglandular caruncular endometrium tissues of control OVX ewes treated with 90% corn oil:10% ethyl alcohol (C), and of OVX ewes treated with E2, P4 or both hormones (P4/E2). Ewes received steroids according to schedules (Table 1Go) designed to produce physiological changes of these hormones during the follicular and luteal phases of the estrous cycle. Values are means ± SEM for the number of animals used per group (n = 4 ewes). *, P < 0.05; **, P < 0.01 compared with values of control OVX ewes.

 
The activity of SOD1 in the glandular tissue significantly decreased after P4 treatment of OVX ewes, compared with that in control OVX ewes (Fig. 4AGo). The activities of SOD2, CAT, GPX, and GSR in the glandular tissue were unaffected by treatment of OVX ewes with P4, as compared with those in control OVX ewes (Fig. 4Go, B–E). The activities of SOD1, CAT and GPX significantly decreased in glandular tissues of OVX ewes that received E2, either alone or in combination with P4, as compared with those in control OVX ewes (Fig. 4Go, A, C, and D). No significant change in the activity of SOD2 was detected in the glandular tissue after treatment of OVX with E2, P4, or both hormones (Fig. 4BGo). The activity of GSR significantly decreased in the glandular tissue after E2 treatment of OVX ewes, either alone or in combination with P4, as compared with that in control OVX ewes (Fig. 4EGo).


Figure 4
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  		FIG. 4. Activities of antioxidant enzymes (A, SOD1; B, SOD2; C, CAT; D, GPX; and E, GSR) in the sheep glandular intercaruncular endometrium tissues of control OVX ewes treated with 90% corn oil: 10% ethyl alcohol (C), and of OVX ewes treated with E2, P4, or both hormones (P4/E2). Ewes received steroids according to schedules (Table 1Go) designed to produce physiological changes of these hormones during the follicular and luteal phases of the estrous cycle. Values are means ± SEM for the number of animals used per group (n = 4 ewes). *, P < 0.05; **, P < 0.01 compared with values of control OVX ewes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our present study is the first to examine the effects of E2 and P4 on key antioxidant enzymatic pathways in the two morphologically and functionally distinct tissues of the sheep endometrium. Because the enzymatic activity is directly responsible for the physiological effect, changes in antioxidant enzymes activities may indicate how an organ or a tissue, such as the endometrium, might respond to the changes in the endocrine environment under in vivo physiological conditions. Criticisms of previous studies investigating the effect of E2 in immature mice (11), and the effect of E2, P4 or both hormones in OVX rats (24) on antioxidant pathways of the uterus is that only SOD or GSR activity was investigated, and that high doses of steroid hormones were used. Treatment of OVX ewes with E2 and P4 (present study) produced the episodic-like pattern of systemic concentrations of these steroid hormones observed during the estrous cycle in intact ewes (16). The preovulatory surge of E2 up-regulates estrogen and P4 receptors expression in the endometrium of all species examined, including sheep (25), thus enabling ovarian steroids to induce well-characterized endometrial cell growth and proliferation (26, 27). The administration of 16 µg E2/d for 2 d (present study) produced a preovulatory surge of this hormone similar to that reported in intact ewes (16). During steroid treatments of OVX ewes, circulating P4 concentrations showed a cyclic pattern and three peaks of E2 occurred on d 2–4, 6–8, and 10–12 (present study), which correspond to follicular E2 waves during the luteal phase in cyclic ewes (16). Therefore, an important strength of our work is that the effects of E2 and P4 treatments on activities of key antioxidant enzymes were observed using an integrative, whole animal model with exposure to physiological steroid hormone concentrations, thus emphasizing the relevance of our findings to normal sheep endometrium function.

Effects of E2 and P4 in vivo on cytoplasmic SOD1 and mitochondrial SOD2 activities in mammalian endometrium have not previously been reported. Interestingly, SOD activity in the uterine tissue of immature mice was lowered by administration of E2, and increased by means of anti-estrogenic treatments in a dose-dependent manner (11). The main limitation of this study was that only total SOD activity was measured and this provided no information of the changes in activity of SOD1 and/or SOD2. In the present study, we showed that SOD1 activity in both the aglandular and glandular endometrial tissues was markedly down-regulated by E2 treatment, and coadministration with P4 did not affect this E2 effect. Treatment with P4 down-regulates SOD1 activity only in the glandular endometrial tissue. P4 or E2 treatment alone or in combination has no effect on SOD2 activity, irrespective of the endometrial tissues examined. Although the SOD1 activity in both endometrial tissues is found to be under the control of E2, appropriate hormonal priming with physiological concentrations of P4 is needed for such action (present study).

The data presented here indicate that E2, but not P4, plays a more important role in the regulation of SOD1 in the endometrium of OVX ewes. In contrast to our in vivo study, it has been shown from in vitro experiments that treatment of human endometrial stromal cells with a synthetic form of P4, medroxyprogesterone acetate (MPA, Provera), for 18 d increase SOD1 and SOD2 activities, whereas E2 has an additive effect only on SOD1 activity (12). However, one should be cautious when drawing conclusions from in vitro studies regarding physiological situations, because culture conditions cannot fully replicate the in vivo environment. In addition, discrepancies in results could be, in part, attributable to differences in the cellular responses induced by different progestins. Indeed, natural P4 and MPA can induce divergent responses. It has been reported that treatment of OVX monkeys with physiological concentrations of E2 and P4 reduced coronary artery vasospasm, whereas MPA, in contrast to P4, negated the protective effect of E2 (28). Furthermore, P4 and 19-norprogesterone, but not MPA, decreased neuronal damage induced by glutamate toxicity in vitro (29, 30).

Steroid control of CAT and GSH-dependent enzyme activities in the mammalian endometrium has not previously been reported. CAT and GPX have a major antioxidant role within cells, because they act to detoxify H2O2 generated by SOD. GSR exerts its antioxidant protective role indirectly by facilitating GSH synthesis and thus supplying GSH for GPX-catalyzed reactions. No changes in CAT, GPX, and GSR activities were detected in the aglandular and glandular endometrial tissue after administration of P4. CAT and GPX activities were decreased in both endometrial tissues after E2 administration, and coadministration with P4 did not affect this E2 effect. The activity of GSR decreased only in the glandular tissue after E2 treatment, either alone or in combination with P4. Our findings provide the first evidence that E2 plays a pivotal role in the regulation of CAT, GPX and GSR activities in the endometrium of OVX ewes.

ROS are generated in the human endometrium and increased in the late secretory phase just before menstruation (7). It is therefore suggested the increase in ROS production may contribute to the shedding of the human endometrium. It has been reported that E2 stimulated the production of H2O2 by supernatant fractions of uteri from ovariectomized rats in a dose-dependent manner, whereas P4 had no significant effect (31). In addition, naturally occurring changes in the concentrations of E2 influenced the endogenous generation of H2O2, as demonstrated by the high rate of production of H2O2 in the uterine supernatant fractions of estrous rats as compared with the diestrous rats (31). E2 has been shown also to regulate O2·– generation by lowering SOD activity in the uterus of ovariectomized mice (32). Together, these findings and the results of our study lead us to consider the possibility that down-regulation of antioxidant enzyme activities by E2 may play a physiological role in later stages of the estrous cycle in sheep.

Very little is known about the effects of steroid hormones in the expression of mRNA encoding the various antioxidant enzyme genes in female reproductive system. The existence of a relationship between the first line of antioxidant scavenging system and ovarian steroids has been demonstrated in the ruminant follicle. In the cow, expression of mRNA encoding SOD1 and SOD2 in granulosa cells of dominant follicles was greater during the midluteal phase than the preovulatory phase, when follicular E2 production is maximum (33). An inverse relationship was found between intrafollicular SOD activity and concentrations of E2 content in the follicles of different sizes from sheep and goats (34). Furthermore, E2 effectively regulates O2·– generation by lowering SOD activity in the uterus of ovariectomized mice (32). Together, these previous studies suggest that E2 may play a role in the down-regulation of SOD expression and activity. It is possible that E2 down-regulates specific isoforms of antioxidant enzymes in the endometrium. In the future, it will be necessary to examine the mRNA expression of SOD and other antioxidant enzymes during the estrous cycle to evaluate potential mechanisms of antioxidant regulation by E2 and other factors.

In conclusion, this study provides the first firm evidence for the role of ovarian steroid hormones in the regulation of the activities of key antioxidant enzyme in the endometrium of female mammals. Our in vivo study has led us to suggest that down regulation of antioxidant enzyme activities by ovarian steroids may be linked to ROS generation and physiological endometrium function in nonpregnant sheep. While these conclusions are applicable to the experiment model used in the present study, changes in the activities of key antioxidant enzymes in the endometrium in relation to the secretion of ovarian hormones throughout the estrous cycle remain to be elucidated. Further studies are also needed to elucidate the mechanisms of the actions of steroid hormones to control endometrial antioxidant enzyme pathways. The findings reported here establish a reference database for future studies of antioxidant adaptive responses in endometrium exposed to disturbed endocrine environment.


   Acknowledgments
 
The authors would like to thank Krawiec Angele, Catherine Mangournet, Sandra Grange, Christine Tozzoli, Cécile Mounioz, and Christian Poirier for their excellent technical assistance. The authors also thank the staff of the sheep sheds of Brouëssy and Jouy-en-Josas for outstanding technical help and sheep management. We are grateful to Dr. Dairena Gaffney (Department of Biochemistry, Glasgow Royal Infirmary, UK) for her comments and carefully reading the manuscript.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online May 29, 2008

Abbreviations: CAT, Catalase; E2, 17β-estradiol; OVX, ovariectomized; GPX, glutathione peroxidase; GSH, glutathione; GSR, glutathione reductase; MPA, medroxyprogesterone acetate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; P4, progesterone; ROS, reactive oxygen species; SOD1, copper, zinc-superoxide dismutase; SOD2, manganese-SOD.

Received February 8, 2008.

Accepted for publication May 20, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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