Estrogen Selectively Up-Regulates the Phospholipid Hydroperoxide Glutathione Peroxidase in the Oviducts
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内分泌学杂志 2005年第6期
Unité de Recherche en Ontogénie et Reproduction (J.L., J.-F.B.), Centre de Recherche du Centre Hospitalier de l’Université Laval, and Centre de Recherche en Biologie de la Reproduction (J.L., J.-F.B.), Université Laval, Québec, Canada G1V 4G2; Département d’Obstétrique et Gynécologie (J.-F.B.), Université Laval, Québec, Canada G1K 7P4; and Department of Plant and Animal Sciences (S.K., L.A.M.), Nova Scotia Agricultural College, Truro, Nova Scotia, Canada B2N 5E3
Address all correspondence and requests for reprints to: Jean-Fran?ois Bilodeau, Unité d’Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Pavillon Centre Hospitalier de l’Université Laval, Local T-1-49, 2705 Boulevard W. Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: jean-francois.bilodeau@CRCHUL.Ulaval.ca.
Abstract
The oviduct plays a crucial role in mammalian reproduction by providing an optimal environment for the final maturation and transport of gametes, fertilization, and early embryonic development. It is now recognized that these reproductive events in vitro can be either negatively or positively affected by reactive oxygen species such as hydrogen peroxide and lipid hydroperoxides. In the current study, we analyzed the expression of the phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx-4), a selenoenzyme that directly reduces membrane-bound lipid hydroperoxides in the bovine oviduct. Using in situ hybridization, we demonstrated that GPx-4 expression is almost restricted to the oviductal luminal epithelium in contrast to GPx-1, which is widely distributed, and GPx-2 and -3, which are mainly detected in the epithelial cells and lamina propria. Interestingly, real-time quantitative RT-PCR analysis showed that GPx-4 expression was highest during the follicular and postovulatory phases. In addition, GPx-4 expression was highest in the isthmus proximal to the dominant follicle during the follicular stage and remained high during the postovulatory period. This increased in expression of GPx-4 corresponded to increased GPx-4 enzymatic activity. Based on intrauterine infusion of estradiol, we determined that the increase in expression and activity of GPx-4 is estrogen mediated. This work clearly demonstrates that GPx-4 gene expression is influenced by the proximity of the dominant follicle in the oviduct in vivo. We propose that GPx-4 has an important role in the physiological control of peroxide tone in the bordering cells of the oviductal lumen.
Introduction
IN MAMMALIAN REPRODUCTION, the oviducts are the site of crucial processes that occur before implantation such as oocyte maturation, sperm storage and capacitation, gamete fusion and the initial stages of embryonic development (1, 2, 3, 4, 5). It is now recognized that reactive oxygen species (ROS: H2O2, O2–·, OH·, NO...) affect gametes and early reproductive events. Indeed, ROS are largely known for their toxic effects on spermatozoa and have been linked to male infertility (6, 7, 8). Excessive ROS production has been associated with sperm morphological defects (9), inhibition of sperm motility (10, 11), fragmentation of sperm DNA (12), and premature capacitation (13). Furthermore, ROS decrease capacity for sperm-oocyte fusion efficiency and severely inhibit embryo development in vitro (14, 15, 16).
On the other hand, low concentrations of ROS positively affect sperm functions (17, 18), binding of sperm to zona pellucida (19), and embryo development in bovine and others mammals (3, 20, 21). Thus, it appears that the oviducts play a crucial role by fine tuning ROS levels to favor the proper condition for gametes, fertilization, and subsequent stages of embryo development. The intracellular and extracellular ROS concentrations are controlled by enzymatic and nonenzymatic defenses. Recently, we described the presence of an elaborate antioxidant defense system in bovine oviductal tissues and fluids (22). Antioxidant genes, especially some glutathione peroxidases (GPx-1, -2, and -3), were differentially expressed along the oviduct. The family of GPx as well as the oviductal catalase are the major enzymes found in the oviduct that are able to metabolize hydrogen peroxide (H2O2) (23, 24). Depending on the concentration, the latter is known to have both beneficial and detrimental effects on gametes and to affect the early reproductive events that take place in the oviducts (3, 6, 19, 25).
Hydrogen peroxide can react with unproperly chelated iron to produce the hydroxyl radical (OH.), which is one of the ROS that can trigger lipid peroxidation and formation of peroxidative intermediates like lipid hydroperoxides (LOOHs) (26). The LOOHs are also produced by the action of lipoxygenases and cyclooxygenases (27, 28). These are relatively long-lived ROS and are associated with cellular membranes (29). Several studies have revealed that, once formed, LOOHs mediate a variety of deleterious processes leading to various cellular dysfunctions (29). LOOHs have been associated with structural perturbations of biomembranes and lipoproteins, cytotoxicity, DNA damage, and disorders such as atherosclerosis, neurodegeneration and cancer (29, 30, 31, 32, 33). Furthermore, lipid peroxidation can have negative effects on sperm morphology and motility, gamete fusion and fertility in mammals (19, 34, 35). In contrast, recent studies revealed that the controlled production of LOOHs may also have beneficial effects for cells and whole organisms (32). Lipid hydroperoxides have been linked to crucial events such as membrane remodeling and protein trafficking (36), cell maturation and differentiation (32), signal transduction (37), prostaglandins synthesis and apoptosis (38, 39). The dynamic expression of the classical cytosolic GPx-1, the gastrointestinal GPx-2, and the extracellular GPx-3 in the bovine oviduct underlines the importance of the control of H2O2 and LOOHs in the female reproductive tract (22). However, without the help of a phospholipase, these enzymes are unable to reduce membrane-bound lipid hydroperoxides. Thus, GPx-1, -2, and -3 alone cannot counteract the numerous effects generated by the peroxidation of membrane lipids.
The phospholipid hydroperoxide glutathione peroxidase (PHGPx) is the fourth member of the GPx family (GPx-4), and is the only known intracellular enzymatic antioxidant that can directly reduce both phospholipids and cholesterol-hydroperoxides located in cell membranes (40, 41) (see reactions 1–4).
Reactions:
This unique enzyme is widely expressed in normal tissues, particularly in the endocrine organs, and is found in the nucleus, mitochondria and cytosol (24). Disruption of the gene in mice results in early embryonic lethality (42, 43). Both direct and indirect evidence indicates that GPx-4 is involved in a variety of cellular mechanisms including signal transduction, differentiation, inflammation, and apoptosis (44, 45). In the testicular tissues of humans, mice, and rats, GPx-4 is detected in the seminiferous epithelium, and its expression is influenced by testosterone during spermatogenesis (46, 47, 48). GPx-4 is considered to play multiple roles in spermatogenesis and to be involved in male fertility (49).
In the oviduct, gametes are exposed to a environment where a large number of lipids, potentially peroxidizable by ROS, are secreted and synthesized (22, 50, 51). The first objective of this study was to characterize the mRNA expression and the enzymatic activity of GPx-4 in the oviduct throughout the estrous cycle of the cow. To better understand the potential roles of GPx in oviduct functions, we also undertook the cellular localization of the expression of GPx-1 to -4 and found that these genes are differentially distributed in oviduct cell types. Importantly, our results reveal estrous cycle-dependent regulation of GPx-4 expression, which is differentially modulated in the ipsilateral and contralateral oviducts by estrogen.
Materials and Methods
Oviducts
Bovine oviducts were transported on ice within 4 h of the animal being killed to the laboratory. Animals that showed anomalies of the genital tract were rejected after examination by a veterinarian. The stage of the estrous cycle was defined by postmortem examination of the ovaries (follicle and corpus luteum), and the oviducts were classified into four groups: postovulatory (d 0–3), mid-luteal (d 10–12), late luteal (d 15–17), and follicular (d 18–20) stages according to the criteria documented by Arosh et al. (52). Oviducts ipsilateral and contralateral to the corpus luteum were analyzed separately. During the follicular phase, the contralateral oviduct is the oviduct opposite to the regressing corpus luteum and proximal to the ovary that contains the dominant follicle. All other cases were excluded. Because of their relatively large size (18–25 cm long), bovine oviducts allow RNA analysis on several specific sections of the oviduct. Thus, the oviducts were dissected on an ice-cold glass plate to remove all blood vessels and cut in three sections (isthmus, ishtmic-ampullary junction, and ampulla). The tissues were frozen in liquid nitrogen and kept at –86 C until analysis.
Treatments
All procedures were performed in accordance to the guidelines of the Canadian Council on Animal Care and were reviewed and approved by the Nova Scotia Agricultural College Animal Care and Committee. Six healthy, sexually mature mixed-breed beef heifers (1.5–3 yr of age, 520 ± 31 kg bodyweight) were randomly assigned to control (BSA, n = 3) or 17?-estradiol (n = 3) intrauterine infusion treatments as previously described. Briefly, animals were treated midcycle with Estrumate* (500 mg cloprostenol, Schering Canada Inc., Pointe-Claire, Québec, Canada) to synchronize estrus. In the morning of d 14 after estrus, each heifer received intrauterine infusions in both horns of BSA (0.1% in saline) or 17?-estradiol (117 ng/dose, Sigma, St. Louis, MO). Treatments were delivered five times at 12-h intervals. The animals were slaughtered 7 h after the last treatment on d 16 of the estrous cycle, and the oviducts were collected and processed as previously described (53).
In situ hybridization
Oviduct segments were fixed overnight in 4% paraformaldehyde and embedded in OCT (Canemco, St. Laurent, Québec, Canada). The in situ hybridization protocol using cryosections was based on the method described by Légaré et al. (54). The following bovine fragments were used as templates for synthesizing digoxygenin (DIG)-RNA probes: a 373-bp GPx-1 fragment (bases 258–631 of GenBank sequence no. x13684); a 584-bp GPx-2 fragment (bases 35–619 of GenBank sequence no. NM002083); 405-bp GPx-3 fragment (corresponding to bases 79–474 of the bovine sequence: GenBank no. L10325); and a 400-bp GPx-4 fragment (from positions 38–438 of the bovine sequence no. AB017534). These fragments were cloned into pGEM-T vectors (Promega, Madison, WI), and sense and antisense probes were transcribed using either SP6 or T7 RNA polymerase and the DIG RNA labeling kit according to manufacturer’s instructions (Roche Diagnostics, Laval, Québec, Canada). Oviduct cryosections were incubated overnight at 42 C with the RNA-labeled probes. Hybridization reactions were detected by immunostaining with alkaline phosphatase-conjugated DIG antibodies (Roche Diagnostics), and the blue signal was visualized using the phosphatase substrate NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt). Neutral red was used to counterstain the cryosections. Three separate experiments were performed on five specimens using eight cryosections per specimen.
Images were acquired in color directly from the stained tissues with a Zeiss Axioskop 2 Plus microscope (Toronto, Ontario, Canada) linked to a digital camera using Spot software (Diagnostics Instruments, Sterling Heights, MI). Relative quantification of the specific blue staining was performed by densitometry analysis using Image Pro software (Carsen Medical Scientific, Markham, Ontario, Canada) as described by Doiron et al. (55). Integrated OD of the blue staining was measured after standard OD calibration and results were expressed in integrated OD units. All data were presented as mean ± SEM of five specimens (three oviduct sections were analyzed for each specimen).
Preparation of RNA and cDNA
The RNA was extracted using TRIzol as described in the manufacturer’s instructions (Invitrogen, Burlington, Ontario, Canada). Four micrograms of total RNA were reverse transcribed with random hexamer primers and the Superscript II reverse transcriptase (Invitrogen). The first-strand cDNA was diluted 20 times in sterile water and used as the template in the quantitative RT-PCR mixture.
Quantitative RT-PCR
Sets of specific primers for GPx-1, GPx-2, Gpx-3, PHGPx, and oviductin were designed based on known bovine sequences (Table 1). Classical PCRs were first conducted to confirm the specificity of primers. As an internal control, 18S rRNA was amplified. The expected PCR products were isolated by agarose gel electrophoresis, eluted, cloned, and sequenced. The plasmids containing the cloned fragments were serially diluted from 500 pg to 5 fg and used as templates in quantitative RT-PCRs to establish the standard curves. The quantitative RT-PCRs were carried out in a LightCycler (Roche Diagnostics). Reactions were performed in a 20-μl reaction mixture containing either 5 μl of diluted cDNA, or plasmid standard, 0.25 μM of each primer, 3 μM of MgCl2, 2 μl of FastStart Master SYBRGreen I mix (Roche Diagnostics) and PCR-grade water up to the final volume. The RT-PCRs were performed as follows: denaturation at 95 C for 10 min followed by 45 cycles of amplification (95 C for 0 sec, annealing temperature for 5 sec, and 72 C for 20 sec) with single acquisition of fluorescence at the end of extension step. The annealing temperatures for each gene were: GPx-1 and -2 (68 C); GPx-3 (62 C); GPx-4 (63 C); oviductin (64 C) and 18S (58 C). After amplification, the samples were slowly heated at 0.1 C/sec from 60–95 C with continuous reading of fluorescence to obtain a melting curve. The specificity of each amplicon was then determined by using the melting curve analysis program of the Lightcycler software. The amplicon of GPx-1 to -4, oviductin and 18S showed only a peak in the analysis. The specificity of the amplified products was confirmed by agarose gel electrophoresis. The quantification analysis of the data were performed by using the LightCycler analysis software. Second-derivative maximum analysis, arithmetic base line adjustment and polynomial calculation were used. The concentration of each gene was calculated by reference to the respective standard curve. Relative gene expression was expressed as a ratio of target gene concentration to housekeeping gene (18S rRNA) concentration. All total RNA samples were reverse transcribed twice. Each cDNA was quantified in duplicate and the average value of each sample was used for quantification.
TABLE 1. Details of primers used for quantitative RT-PCR
Activity assay for PHGPx activity
The enzymatic activity was measured by a coupled spectrophotometric enzymatic assay using glutathione, glutathione reductase, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and phosphatidylcholine hydroperoxides (PCOOHs), and was based on methods described by Roveri et al. (56) (see reactions 1–4). The GPx-4 substrate (PCOOH) was prepared by enzymatic peroxidation of phosphatidylcholine using soybean lipoxygenase type IV (Sigma-Aldrich, Oakville, Ontario, Canada). Briefly, a mixture containing 0.3 mM of phosphatidylcholine, 0.2 M Tris-HCl (pH 8.8), 3 mM sodium deoxycholate, and 0.7 mg soybean lipoxygenase type IV was stirred at room temperature for 30 min. The reaction was further loaded onto a Bond Elut Jr C18 column (Varian Inc., Palo Alto, CA), previously equilibrated with 4 ml of methanol and 40 ml of water. The column was washed with 220 ml of water, and the PCOOH was eluted with 3 ml methanol to a final concentration of 1.6 μM. The reaction mixture for enzymatic assays contained the following final concentrations: 0.1 M (KH2PO4/K2HPO4) (pH 7.8), 1 mM EDTA, 1.5 U/ml glutathione reductase (Roche Diagnostics), 3 mM GSH, and 200 μg of oviductal proteins. This mixture was incubate at 37 C for 2 min, then 0.2 mM of NADPH was added and the nonspecific NADPH oxidation rate was recorded at 340 nm for 3 min using a microplate florescence reader FL600 (Bio-Tek, Winooski, VT). The enzymatic reaction was started by the addition of 160 μM of PCOOH and the NADPH oxidation was monitored for 5 min to generate the kinetic curve. Activity was calculated by subtracting the nonspecific NADPH oxidation rate from the observed rate after substrate addition. The specific enzymatic activity is expressed as milliunits per milligram total cell protein; one unit of enzyme activity is defined by the oxidation of 1 μM of NADPH/min.
Statistical analysis
Values shown in the text and tables are mean ± SEM. All data were normally distributed and passed equal variance testing. Model variables included oviduct section (isthmus, ishtmic-ampullary junction and ampulla), oviduct side (ipsilateral vs. contralateral to the corpus luteum), and stage of the estrous cycle (d 0–3, 10–12, 15–17, and 18–20). Main effects of each variables and interactions between these variables were determined. The experiments were analyzed with the general linear model of SPSS 10.0 for Windows (SPSS Inc., Chicago, IL). Multiple means were compared by ANOVA and when a significant effect was obtained, the difference between means was determined by Duncan multiple range test. A P value of less than 0.05 was considered statistically significant.
Results
GPx-1, -2, -3, and -4 are expressed in different oviductal cell types
We localized the cellular mRNA expression of the GPx-1, -2, -3, and -4 along the bovine oviduct using in situ hybridization. Our results revealed a cell-specific distribution between cell types for each of the GPx mRNAs in the different oviduct segments. The cellular distribution of GPx-1, -2, -3, and -4 in isthmus, ishtmic-ampullary junction and ampulla is consistent throughout the estrous cycle (data not shown). The classic or cytosolic glutathione peroxidase, GPx-1, is strongly expressed in all oviduct segments and all oviductal cell types (Fig. 1, A–C). Relative quantification revealed that GPx-1 was mostly expressed in the lamina propria and smooth muscle cells of the isthmic-ampullary junction and the ampulla sections (Table 2). Furthermore, the patterns of expression for the gastrointestinal GPx-2 and the extracellular GPx-3 also differed in the three oviduct segments (Fig. 1, D–I; Table 2). Indeed, GPx-2 was found in the oviductal epithelium and the lamina propria, the connective tissue containing blood vessels and free glands blending with the submucosa, of all oviduct sections. Furthermore, positive staining for GPx-2 was also seen in the smooth muscle layer of the ampulla (Fig. 1F; Table 2). In the isthmus and the ishtmic-ampullary junction, GPx-3 expression is mainly detected within the lamina propria (Fig. 1, G and H). In the ampulla, GPx-3 expression is also observed in the single layer epithelium and in the thin smooth muscle layer (Fig. 1I; Table 2). In comparison to GPx-1 to -3, GPx-4 has the most exclusive expression pattern, being restricted to the epithelium in the three oviduct segments (Fig. 1, J–L). Indeed, the intense GPx-4 signal found in the epithelium along the oviduct contrast with the very weak blue staining detected in the circular smooth muscle layer (Table 2).
FIG. 1. In situ localization of GPx transcripts along the bovine oviduct. Transversal cryosections of isthmus, ishtmic-ampullary junction, and ampulla segments were probed with DIG-labeled antisense GPx-1 to -4 RNA probes. GPx mRNAs were detected by immunostaining using alkaline phosphatase-labeled anti-DIG, which appear as a blue staining after incubation with NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) substrate. GPx-1 was ubiquitously expressed in the three sections of the oviduct (A–C). GPx-2 was strongly expressed in the epithelium and was also detected in the lamina propria (D–F) and in the smooth muscle layer of the ampulla (F). GPx-3 expression was mainly located in the lamina propria of the isthmus (G), in the lamina propria, the epithelium and the smooth muscle layer in other segments (H and I). GPx-4 mRNA was primarily detected in oviductal epithelium (J–L). Sense probes were used as negative control (M–O, representative controls). Sections were counterstained with neutral red to visualize cell nuclei. Sense probes were used as negative controls and showed that a nonspecific signal is observed only in the serosa region, and thus was not considered in our interpretation of the results. EP, Epithelial cells; L, oviduct lumen; LP, lamina propria; SE, serosa; SM, smooth muscle cells. Final magnification, x100 and x400.
TABLE 2. Relative quantification of GPx-1 to -4 in the oviduct following in situ hybridization studies (see Fig. 1).
Modulation of GPx-4 expression in the oviduct throughout the estrous cycle
The overall oviductal expression pattern of GPx-4 was determined for the four different stages of the estrous cycle by quantitative RT-PCR. Importantly, GPx-4 expression was highest during the follicular (d 18–20) and postovulatory (d 0–3) stages of the estrous cycle in comparison to the mid-luteal (d 10–12) and the late luteal (d 15–17) stages (Fig. 2, A). To further investigate the regional expression of GPx-4 along the oviduct, three equidistant sections of the oviduct were examined at the same four stages of the cycle (Fig. 2, B–D). Analysis revealed that GPx-4 mRNA expression throughout the estrous cycle varies between the different oviduct sections (Fig. 2, B–D). GPx-4 expression in the isthmus follows the trend observed for the whole oviduct with the highest levels of expression during the d 18–20 and 0–3 (Fig. 2B). In the ishtmic-ampullary junction, GPx-4 expression increases during the follicular and postovulatory stages, but no significant variation is observed between the postovulatory and the late-luteal stages (Fig. 2C). In the ampulla, homogenous GPx-4 expression is present during the postovulatory, the mid-luteal, and the late-luteal stages (Fig. 2D).
FIG. 2. GPx-4 mRNA expression in the bovine oviduct throughout the estrous cycle. The mRNA levels were measured by real-time quantitative PCR. GPx-4 mRNA expression was quantified in the whole oviduct (A), in the isthmus (B), the ishtmic-ampullary junction (C), and the ampulla (D) segments of the oviduct. Expression of GPx-4 at the postovulatory (d 0–3), the mid-luteal (d 10–12), the late-luteal (d 15–17) and follicular (d 18–20) stages of the estrous cycle was compared for each section. The 18S rRNA was used as an internal standard and results are expressed as a ratio of GPx-4/18S. Data are means ± SEM of five animals. Means with different designations (*, ) are significantly (P < 0.05) different from each other (Duncan’s test).
Expression of GPx-4 in the ipsilateral and contralateral oviducts during the estrous cycle
To determine whether the expression of GPx-4 was affected by the proximity of the cycling ovary, oviducts ipsilateral and contralateral to the corpus luteum were analyzed (Fig. 3, A–C). We examined ovaries to select the oviducts from cows characterized only by a change of ovulation side between two consecutive estrous cycles. We observed that in the isthmus and the ishtmic-ampullary junction, GPx-4 expression was highest in the oviduct proximal to the dominant follicle during the follicular phase (Fig. 3, A and B). During the postovulary stage, the GPx-4 mRNA expression is up-regulated in the oviduct ipsilateral to the ovulation side in these sections. Interestingly, GPx-4 expression was not modulated between the ipsilateral and the contralateral oviducts in the ampulla area during the follicular and the postovulatory stages of the estrous cycle (Fig. 3C).
FIG. 3. Differential GPx-4 mRNA expression between the ipsilateral and contralateral oviducts. The mRNA levels were measured by real-time quantitative PCR. The 18S rRNA was used as an internal standard and results are expressed as a ratio of GPx-4/18S. GPx-4 mRNA levels in the ipsilateral () and contralateral () oviducts at the postovulatory (d 0–3) and follicular (d 18–20) stages of the estrous cycle were quantified in the isthmus (A), the ishtmic-ampullary junction (B) and the ampulla (C). Data are means ± SEM of eight animals. Means with designations (*) are significantly different at P < 0.05 (Duncan’s test).
GPx-4 enzymatic activity in the ipsilateral and contralateral oviducts
To determine whether GPx-4 expression patterns paralleled function, its enzymatic activity was measured in whole oviducts at same three stages of the estrous cycle. Our results indicated that the GPx-4 activity was modulated throughout the estrous cycle because significant changes were observed between the three stages of the cycle (Fig. 4A). The highest GPx-4 activity was observed during the postovulatory stage, and the lowest activity was measured during the mid-luteal phase. Detailed analysis of GPx-4 activity in isthmus sections from the ipsilateral and contralateral oviducts revealed that the specific activity was higher in the ipsilateral isthmus during the postovulatory stage, and in the contralateral isthmus during the follicular stage (Fig. 4B). This was consistent with mRNA expression (Fig. 3A). GPx-4 activity was similar in isthmus from both sides at the mid-luteal stage (d 10–12), and no significant variation in GPx-4 activity was also observed between the ipsilateral and the contralateral ampulla sections at all stages of the estrous cycle (Fig. 4C). The results for the enzymatic activities are therefore in accordance with mRNA expression. Interestingly, GPx-4 expression and activity are up-regulated during the estrogen-high phases of the estrous cycle (Figs. 2A and Fig. 4A).
FIG. 4. Analysis of GPx-4-specific activity in the bovine oviduct throughout the estrous cycle. Enzymatic activity was measured by a coupled enzymatic assay at 340 nm. The phosphatidylcholine hydroperoxide was used as substrate. One unit of GPx-4 activity is defined as the amount of protein required to oxidize 1 μM of NADPH/min. A, GPx-4 activity was measured in the whole oviduct at the post ovulatory (d 0–3), mid-luteal (d 10–12) and follicular (d 18–20) stages of the estrous cycle. Data are means ± SEM of eight animals. B and C, Representation of GPx-4 activity in the isthmus and the ampulla segments from ipsilateral () and contralateral () oviducts at the three stages of the estrous cycle. Data are means ± SEM of three animals. Means with different designations (*, ) are significantly (P < 0.05) different from each other (Duncan’s test).
Effects of 17?-estradiol on GPx expression in the oviducts in vivo
Because GPx-4 expression appeared to be regulated during the estrous cycle and influenced by the proximity of the dominant follicle, heifers were treated with intrauterine infusions of 17?-estradiol to test the hypothesis that estradiol up-regulates GPx-4 expression in the bovine oviduct. The mRNA expression of the oviductin, an oviduct-specific glycoprotein known to be regulated by estradiol, was measured by quantitative RT-PCR to ensure estrogen exposure was effective. Estrogen treatment increased expression of oviductin 3-fold in the isthmus and the ampulla (Fig. 5A). Confirming estrous cycle modulated expression of GPx-4, expression was 2-fold higher in the oviducts of cows treated with uterine infusions of 17?-estradiol (Fig. 5E). The effects of the treatment on the transcription of GPx-1 to -3 also were investigated, but no significant variations of the mRNA levels were observed (Fig. 5, B–D).
FIG. 5. Modulation of oviductal GPx-4 expression by 17?-estradiol treatment. The mRNA levels were measured by real-time quantitative PCR (LightCycler). The 18S rRNA was used as an internal standard and results are expressed as a ratio of GPx-4/18S. Cows received either uterine infusion of saline control ( ) or 17?-estradiol () at d 14 of the estrous cycle. The mRNA levels of each gene were quantified in the isthmus and ampulla segments of the oviducts from both groups. A, Oviductin. B, GPx-1. C, GPx-2. D, GPx-3. E, GPx-4. Data are means ± SEM of three animals. Means with designations (*) are significantly different at P < 0.05 (Duncan’s test).
Discussion
We have previously reported that the classical intracellular GPx-1, the gastrointestinal GPx-2, and the extracellular GPx-3 were expressed differentially along the oviduct, in addition to being modulated during the estrous cycle (22). Here, we performed the cellular localization of these genes within the oviduct and found unique expression patterns for each GPx. The classical GPx-1 was ubiquitously expressed in all oviductal cell types. The results for GPx-1 were expected because this gene is known to be expressed in almost all cells of mammalian tissues (24). GPx-1 is involved in the detoxification of various intracellular free hydroperoxides. We also demonstrated that GPx-2 was strongly expressed in the epithelial cells of the oviduct. Similarly, GPx-2 was detected in the mucosal epithelium of the gastrointestinal tract (57). In the intestine, GPx-2 is believed to be involved in the neutralization of ingested LOOHs and in the suppression of inflammation (57, 58). The localization of GPx-3 expression in the lamina propria and in luminal epithelial cells of the ampulla is consistent with a role in regulation of extracellular hydroperoxide levels in the oviductal connective tissues and lumen. It is known that extracellular LOOHs can negatively affect the integrity of sperm and even prevent gamete fusion in vitro (7, 18, 25). GPx-3 is expressed at significant levels in the male genital tract, and the protein is found in the lumen of the caput epididymis (59). Because a sperm reservoir in the initial segment of the oviduct has been uncovered in several mammalian species, it is reasonable to believe that the presence of GPx-3 in this specific site of the oviduct must be important to prevent deleterious effects of luminal LOOHs on male gamete integrity (5). Thus, the cellular localization as well as the modulation of the expression of GPx-1, -2, and -3 suggests that these enzymes may be important regulators of free hydroperoxides levels in the oviduct.
This is the first report of GPx-4 localization in the oviduct. Strikingly, it was mainly expressed in the oviductal epithelium placing it close to gametes found in the lumen or bound to membrane during the follicular and the postovulatory stages of the estrous cycle. This discovery is of high importance because GPx-4 is the only GPx family member with the ability to reduce lipid hydroperoxides bound to cell membranes (24). Lipid hydroperoxides have high potential to affect key oviduct functions such as gamete fusion and/or prostaglandin synthesis (19, 32), so, potential mitigation of their effects by GPx-4 could be essential to fertility.
We observed a direct relationship between the mRNA expression and the specific enzymatic activity for GPx-4. Thus, the mRNA and enzymatic activity levels follow exactly the same trend throughout the estrous cycle as well as in between the ipsilateral and contralateral oviducts. Our data clearly revealed that GPx-4 expression in the oviduct is affected by the proximity of the cycling ovary. Indeed, we reported that GPx-4 is mostly expressed in the oviduct contralateral to the regressing corpus luteum during the follicular phase and in the oviduct ipsilateral to the ovulation site during the postovulatory stage. This latter observation is in concordance with the results of a recent transcriptomics study performed on bovine oviductal epithelium. Indeed, this study showed that GPx-4 was mostly expressed in the ipsilateral oviductal epithelium at d 3.5 after standing heat by using subtractive hybridization and cDNA array hybridization techniques (60). Although this experiment was performed only at the postovulatory stage, it was proposed that the expression of GPx-4 in the ipsilateral oviduct may be up-regulated in response to the presence of the nonfertilized oocyte or due to signals from the active ovary. Our current observations at three stages of the estrous cycle demonstrate that the expression of GPx-4 is up-regulated in the contralateral oviduct during the follicular phase, thus before the release of the oocyte from the dominant follicle. This strongly suggests that GPx-4 expression in the oviductal epithelium is regulated by a signal from the active ovary rather than in response to the presence of the oocyte.
The mammalian oviduct is known to be under the influence of local steroids, especially the estrogens (61, 62). The estrogens are known to induce the transcription of specific genes in the mammalian oviduct (1, 63, 64, 65). Estrogens regulate the proliferation and differentiation of oviductal epithelium and are probably important for oviductal contraction and secretion (65, 66, 67, 68). The highest concentration of estradiol in the bovine oviduct occurs at the end of the estrous cycle during the follicular phase. Furthermore, estradiol levels are higher in the oviduct located near the dominant follicle, suggesting that the estradiol is locally delivered to the oviduct by this follicle (66). Consistent with estrogen regulation, our analysis clearly demonstrated that the higher GPx-4 expression in the oviduct occurs during the maturation of the dominant follicle and the postovulatory period, whereas GPx-4 expression is at its lowest in the ipsilateral and contralateral oviducts during the luteal phase (Fig. 5).
This prompted us to analyze the in vivo effects of the estradiol on the GPx-4 mRNA expression in the bovine oviduct. Here, we provide the first in vivo demonstration that GPx-4 is up-regulated by estradiol in the female reproductive tract. We found that GPx-4 is positively regulated by estradiol in both the isthmic and ampullary regions of the bovine oviduct. Recently, it was reported that estradiol could also increase GPx-4 mRNA expression in male reproductive organs in the rat (69). The effects of estrogens on antioxidant enzymes have been investigated in others tissues. Recently, it was shown that the estradiol induces manganese superoxide dismutases and extracellular superoxide dismutase in vascular smooth muscle cells (70). Moreover, further studies have revealed that total GPx activity was stimulated by the estrogens in human endometrium and in the rat liver (71, 72). Taken together, these observations suggest that estrogens may regulate the oviductal antioxidative pathway by positively affecting antioxidant defense mechanism. We showed that the estradiol induced GPx-4 expression, but the expression of the GPx-1, -2, and -3 remained unchanged. GPx-4 is considered to be the main line of enzymatic defense against oxidative biomembrane destruction and is thought to be of outstanding importance (40, 73). We propose that the stimulation of GPx-4 by estrogens enhance the protection of the oviductal epithelial cell membrane during the follicular and postovulatory stages.
The cellular mechanisms that mediate the actions of estrogen on GPx-4 expression will need further investigation, but several estrogen-responsive elements were located within the 5'-untranslated region of porcine GPx-4 (74). The ERs and ? are expressed in the bovine oviductal cells at all stages of the estrous cycle (75). Interestingly, ER is up-regulated in the isthmus and the isthmic-ampullary junction during the follicular stage, but expression in the ampulla remains unchanged throughout the estrous cycle. The comparison of GPx-4 mRNA expression between the luteal and the periovulatory periods of the estrous cycle also revealed that modulation of GPx-4 is higher in the isthmus than in the ampulla. Furthermore, Gpx-4 is not differentially expressed between the ipsilateral and contralateral oviducts at the ampulla level during the follicular and the postovulatory stages. These results suggest that during the estrous cycle the effects of the estradiol on GPx-4 expression mainly occur within the isthmus and the ishtmic-ampullary junction. The periovulatory period is characterized by an increase in the synthesis and secretion of lipids and others molecules by the bovine oviduct epithelial (50, 76). Thus, the high levels of GPx-4 activity throughout this phase may prevent deleterious effects of lipid peroxidation on oviductal epithelial cell and could have important consequences to these cells physiological functions.
In summary, we have reported that GPx-4 expression and enzymatic activity in the bovine oviduct are modulated in the estrous cycle in addition to being differentially regulated between the ipsilateral and contralateral oviducts. This is the first time that the expression of a specific gene is shown to be regulated by the proximity of the dominant follicle in mammalian oviduct in vivo. Furthermore, despite that it is becoming clear that GPx-4 plays multiples roles in cellular functions, almost nothing is known about the mechanisms of control of GPx-4 expression by extracellular signals. Here, we showed that GPx-4 expression was up-regulated by estradiol in the oviduct and provided the first in vivo demonstration of GPx-4 regulation by endocrine hormones in mammalian cells. Moreover, to support this result, we have established a relationship between GPx-4 expression pattern and estradiol physiological distribution in the oviducts throughout the estrous cycle. The analysis of the role of GPx-4 in oviductal function as well as the characterization of the mechanisms of GPx-4 regulation by estrogen will be important subjects for future investigations.
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Address all correspondence and requests for reprints to: Jean-Fran?ois Bilodeau, Unité d’Ontogénie et Reproduction, Centre Hospitalier Universitaire de Québec, Pavillon Centre Hospitalier de l’Université Laval, Local T-1-49, 2705 Boulevard W. Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail: jean-francois.bilodeau@CRCHUL.Ulaval.ca.
Abstract
The oviduct plays a crucial role in mammalian reproduction by providing an optimal environment for the final maturation and transport of gametes, fertilization, and early embryonic development. It is now recognized that these reproductive events in vitro can be either negatively or positively affected by reactive oxygen species such as hydrogen peroxide and lipid hydroperoxides. In the current study, we analyzed the expression of the phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx-4), a selenoenzyme that directly reduces membrane-bound lipid hydroperoxides in the bovine oviduct. Using in situ hybridization, we demonstrated that GPx-4 expression is almost restricted to the oviductal luminal epithelium in contrast to GPx-1, which is widely distributed, and GPx-2 and -3, which are mainly detected in the epithelial cells and lamina propria. Interestingly, real-time quantitative RT-PCR analysis showed that GPx-4 expression was highest during the follicular and postovulatory phases. In addition, GPx-4 expression was highest in the isthmus proximal to the dominant follicle during the follicular stage and remained high during the postovulatory period. This increased in expression of GPx-4 corresponded to increased GPx-4 enzymatic activity. Based on intrauterine infusion of estradiol, we determined that the increase in expression and activity of GPx-4 is estrogen mediated. This work clearly demonstrates that GPx-4 gene expression is influenced by the proximity of the dominant follicle in the oviduct in vivo. We propose that GPx-4 has an important role in the physiological control of peroxide tone in the bordering cells of the oviductal lumen.
Introduction
IN MAMMALIAN REPRODUCTION, the oviducts are the site of crucial processes that occur before implantation such as oocyte maturation, sperm storage and capacitation, gamete fusion and the initial stages of embryonic development (1, 2, 3, 4, 5). It is now recognized that reactive oxygen species (ROS: H2O2, O2–·, OH·, NO...) affect gametes and early reproductive events. Indeed, ROS are largely known for their toxic effects on spermatozoa and have been linked to male infertility (6, 7, 8). Excessive ROS production has been associated with sperm morphological defects (9), inhibition of sperm motility (10, 11), fragmentation of sperm DNA (12), and premature capacitation (13). Furthermore, ROS decrease capacity for sperm-oocyte fusion efficiency and severely inhibit embryo development in vitro (14, 15, 16).
On the other hand, low concentrations of ROS positively affect sperm functions (17, 18), binding of sperm to zona pellucida (19), and embryo development in bovine and others mammals (3, 20, 21). Thus, it appears that the oviducts play a crucial role by fine tuning ROS levels to favor the proper condition for gametes, fertilization, and subsequent stages of embryo development. The intracellular and extracellular ROS concentrations are controlled by enzymatic and nonenzymatic defenses. Recently, we described the presence of an elaborate antioxidant defense system in bovine oviductal tissues and fluids (22). Antioxidant genes, especially some glutathione peroxidases (GPx-1, -2, and -3), were differentially expressed along the oviduct. The family of GPx as well as the oviductal catalase are the major enzymes found in the oviduct that are able to metabolize hydrogen peroxide (H2O2) (23, 24). Depending on the concentration, the latter is known to have both beneficial and detrimental effects on gametes and to affect the early reproductive events that take place in the oviducts (3, 6, 19, 25).
Hydrogen peroxide can react with unproperly chelated iron to produce the hydroxyl radical (OH.), which is one of the ROS that can trigger lipid peroxidation and formation of peroxidative intermediates like lipid hydroperoxides (LOOHs) (26). The LOOHs are also produced by the action of lipoxygenases and cyclooxygenases (27, 28). These are relatively long-lived ROS and are associated with cellular membranes (29). Several studies have revealed that, once formed, LOOHs mediate a variety of deleterious processes leading to various cellular dysfunctions (29). LOOHs have been associated with structural perturbations of biomembranes and lipoproteins, cytotoxicity, DNA damage, and disorders such as atherosclerosis, neurodegeneration and cancer (29, 30, 31, 32, 33). Furthermore, lipid peroxidation can have negative effects on sperm morphology and motility, gamete fusion and fertility in mammals (19, 34, 35). In contrast, recent studies revealed that the controlled production of LOOHs may also have beneficial effects for cells and whole organisms (32). Lipid hydroperoxides have been linked to crucial events such as membrane remodeling and protein trafficking (36), cell maturation and differentiation (32), signal transduction (37), prostaglandins synthesis and apoptosis (38, 39). The dynamic expression of the classical cytosolic GPx-1, the gastrointestinal GPx-2, and the extracellular GPx-3 in the bovine oviduct underlines the importance of the control of H2O2 and LOOHs in the female reproductive tract (22). However, without the help of a phospholipase, these enzymes are unable to reduce membrane-bound lipid hydroperoxides. Thus, GPx-1, -2, and -3 alone cannot counteract the numerous effects generated by the peroxidation of membrane lipids.
The phospholipid hydroperoxide glutathione peroxidase (PHGPx) is the fourth member of the GPx family (GPx-4), and is the only known intracellular enzymatic antioxidant that can directly reduce both phospholipids and cholesterol-hydroperoxides located in cell membranes (40, 41) (see reactions 1–4).
Reactions:
This unique enzyme is widely expressed in normal tissues, particularly in the endocrine organs, and is found in the nucleus, mitochondria and cytosol (24). Disruption of the gene in mice results in early embryonic lethality (42, 43). Both direct and indirect evidence indicates that GPx-4 is involved in a variety of cellular mechanisms including signal transduction, differentiation, inflammation, and apoptosis (44, 45). In the testicular tissues of humans, mice, and rats, GPx-4 is detected in the seminiferous epithelium, and its expression is influenced by testosterone during spermatogenesis (46, 47, 48). GPx-4 is considered to play multiple roles in spermatogenesis and to be involved in male fertility (49).
In the oviduct, gametes are exposed to a environment where a large number of lipids, potentially peroxidizable by ROS, are secreted and synthesized (22, 50, 51). The first objective of this study was to characterize the mRNA expression and the enzymatic activity of GPx-4 in the oviduct throughout the estrous cycle of the cow. To better understand the potential roles of GPx in oviduct functions, we also undertook the cellular localization of the expression of GPx-1 to -4 and found that these genes are differentially distributed in oviduct cell types. Importantly, our results reveal estrous cycle-dependent regulation of GPx-4 expression, which is differentially modulated in the ipsilateral and contralateral oviducts by estrogen.
Materials and Methods
Oviducts
Bovine oviducts were transported on ice within 4 h of the animal being killed to the laboratory. Animals that showed anomalies of the genital tract were rejected after examination by a veterinarian. The stage of the estrous cycle was defined by postmortem examination of the ovaries (follicle and corpus luteum), and the oviducts were classified into four groups: postovulatory (d 0–3), mid-luteal (d 10–12), late luteal (d 15–17), and follicular (d 18–20) stages according to the criteria documented by Arosh et al. (52). Oviducts ipsilateral and contralateral to the corpus luteum were analyzed separately. During the follicular phase, the contralateral oviduct is the oviduct opposite to the regressing corpus luteum and proximal to the ovary that contains the dominant follicle. All other cases were excluded. Because of their relatively large size (18–25 cm long), bovine oviducts allow RNA analysis on several specific sections of the oviduct. Thus, the oviducts were dissected on an ice-cold glass plate to remove all blood vessels and cut in three sections (isthmus, ishtmic-ampullary junction, and ampulla). The tissues were frozen in liquid nitrogen and kept at –86 C until analysis.
Treatments
All procedures were performed in accordance to the guidelines of the Canadian Council on Animal Care and were reviewed and approved by the Nova Scotia Agricultural College Animal Care and Committee. Six healthy, sexually mature mixed-breed beef heifers (1.5–3 yr of age, 520 ± 31 kg bodyweight) were randomly assigned to control (BSA, n = 3) or 17?-estradiol (n = 3) intrauterine infusion treatments as previously described. Briefly, animals were treated midcycle with Estrumate* (500 mg cloprostenol, Schering Canada Inc., Pointe-Claire, Québec, Canada) to synchronize estrus. In the morning of d 14 after estrus, each heifer received intrauterine infusions in both horns of BSA (0.1% in saline) or 17?-estradiol (117 ng/dose, Sigma, St. Louis, MO). Treatments were delivered five times at 12-h intervals. The animals were slaughtered 7 h after the last treatment on d 16 of the estrous cycle, and the oviducts were collected and processed as previously described (53).
In situ hybridization
Oviduct segments were fixed overnight in 4% paraformaldehyde and embedded in OCT (Canemco, St. Laurent, Québec, Canada). The in situ hybridization protocol using cryosections was based on the method described by Légaré et al. (54). The following bovine fragments were used as templates for synthesizing digoxygenin (DIG)-RNA probes: a 373-bp GPx-1 fragment (bases 258–631 of GenBank sequence no. x13684); a 584-bp GPx-2 fragment (bases 35–619 of GenBank sequence no. NM002083); 405-bp GPx-3 fragment (corresponding to bases 79–474 of the bovine sequence: GenBank no. L10325); and a 400-bp GPx-4 fragment (from positions 38–438 of the bovine sequence no. AB017534). These fragments were cloned into pGEM-T vectors (Promega, Madison, WI), and sense and antisense probes were transcribed using either SP6 or T7 RNA polymerase and the DIG RNA labeling kit according to manufacturer’s instructions (Roche Diagnostics, Laval, Québec, Canada). Oviduct cryosections were incubated overnight at 42 C with the RNA-labeled probes. Hybridization reactions were detected by immunostaining with alkaline phosphatase-conjugated DIG antibodies (Roche Diagnostics), and the blue signal was visualized using the phosphatase substrate NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt). Neutral red was used to counterstain the cryosections. Three separate experiments were performed on five specimens using eight cryosections per specimen.
Images were acquired in color directly from the stained tissues with a Zeiss Axioskop 2 Plus microscope (Toronto, Ontario, Canada) linked to a digital camera using Spot software (Diagnostics Instruments, Sterling Heights, MI). Relative quantification of the specific blue staining was performed by densitometry analysis using Image Pro software (Carsen Medical Scientific, Markham, Ontario, Canada) as described by Doiron et al. (55). Integrated OD of the blue staining was measured after standard OD calibration and results were expressed in integrated OD units. All data were presented as mean ± SEM of five specimens (three oviduct sections were analyzed for each specimen).
Preparation of RNA and cDNA
The RNA was extracted using TRIzol as described in the manufacturer’s instructions (Invitrogen, Burlington, Ontario, Canada). Four micrograms of total RNA were reverse transcribed with random hexamer primers and the Superscript II reverse transcriptase (Invitrogen). The first-strand cDNA was diluted 20 times in sterile water and used as the template in the quantitative RT-PCR mixture.
Quantitative RT-PCR
Sets of specific primers for GPx-1, GPx-2, Gpx-3, PHGPx, and oviductin were designed based on known bovine sequences (Table 1). Classical PCRs were first conducted to confirm the specificity of primers. As an internal control, 18S rRNA was amplified. The expected PCR products were isolated by agarose gel electrophoresis, eluted, cloned, and sequenced. The plasmids containing the cloned fragments were serially diluted from 500 pg to 5 fg and used as templates in quantitative RT-PCRs to establish the standard curves. The quantitative RT-PCRs were carried out in a LightCycler (Roche Diagnostics). Reactions were performed in a 20-μl reaction mixture containing either 5 μl of diluted cDNA, or plasmid standard, 0.25 μM of each primer, 3 μM of MgCl2, 2 μl of FastStart Master SYBRGreen I mix (Roche Diagnostics) and PCR-grade water up to the final volume. The RT-PCRs were performed as follows: denaturation at 95 C for 10 min followed by 45 cycles of amplification (95 C for 0 sec, annealing temperature for 5 sec, and 72 C for 20 sec) with single acquisition of fluorescence at the end of extension step. The annealing temperatures for each gene were: GPx-1 and -2 (68 C); GPx-3 (62 C); GPx-4 (63 C); oviductin (64 C) and 18S (58 C). After amplification, the samples were slowly heated at 0.1 C/sec from 60–95 C with continuous reading of fluorescence to obtain a melting curve. The specificity of each amplicon was then determined by using the melting curve analysis program of the Lightcycler software. The amplicon of GPx-1 to -4, oviductin and 18S showed only a peak in the analysis. The specificity of the amplified products was confirmed by agarose gel electrophoresis. The quantification analysis of the data were performed by using the LightCycler analysis software. Second-derivative maximum analysis, arithmetic base line adjustment and polynomial calculation were used. The concentration of each gene was calculated by reference to the respective standard curve. Relative gene expression was expressed as a ratio of target gene concentration to housekeeping gene (18S rRNA) concentration. All total RNA samples were reverse transcribed twice. Each cDNA was quantified in duplicate and the average value of each sample was used for quantification.
TABLE 1. Details of primers used for quantitative RT-PCR
Activity assay for PHGPx activity
The enzymatic activity was measured by a coupled spectrophotometric enzymatic assay using glutathione, glutathione reductase, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and phosphatidylcholine hydroperoxides (PCOOHs), and was based on methods described by Roveri et al. (56) (see reactions 1–4). The GPx-4 substrate (PCOOH) was prepared by enzymatic peroxidation of phosphatidylcholine using soybean lipoxygenase type IV (Sigma-Aldrich, Oakville, Ontario, Canada). Briefly, a mixture containing 0.3 mM of phosphatidylcholine, 0.2 M Tris-HCl (pH 8.8), 3 mM sodium deoxycholate, and 0.7 mg soybean lipoxygenase type IV was stirred at room temperature for 30 min. The reaction was further loaded onto a Bond Elut Jr C18 column (Varian Inc., Palo Alto, CA), previously equilibrated with 4 ml of methanol and 40 ml of water. The column was washed with 220 ml of water, and the PCOOH was eluted with 3 ml methanol to a final concentration of 1.6 μM. The reaction mixture for enzymatic assays contained the following final concentrations: 0.1 M (KH2PO4/K2HPO4) (pH 7.8), 1 mM EDTA, 1.5 U/ml glutathione reductase (Roche Diagnostics), 3 mM GSH, and 200 μg of oviductal proteins. This mixture was incubate at 37 C for 2 min, then 0.2 mM of NADPH was added and the nonspecific NADPH oxidation rate was recorded at 340 nm for 3 min using a microplate florescence reader FL600 (Bio-Tek, Winooski, VT). The enzymatic reaction was started by the addition of 160 μM of PCOOH and the NADPH oxidation was monitored for 5 min to generate the kinetic curve. Activity was calculated by subtracting the nonspecific NADPH oxidation rate from the observed rate after substrate addition. The specific enzymatic activity is expressed as milliunits per milligram total cell protein; one unit of enzyme activity is defined by the oxidation of 1 μM of NADPH/min.
Statistical analysis
Values shown in the text and tables are mean ± SEM. All data were normally distributed and passed equal variance testing. Model variables included oviduct section (isthmus, ishtmic-ampullary junction and ampulla), oviduct side (ipsilateral vs. contralateral to the corpus luteum), and stage of the estrous cycle (d 0–3, 10–12, 15–17, and 18–20). Main effects of each variables and interactions between these variables were determined. The experiments were analyzed with the general linear model of SPSS 10.0 for Windows (SPSS Inc., Chicago, IL). Multiple means were compared by ANOVA and when a significant effect was obtained, the difference between means was determined by Duncan multiple range test. A P value of less than 0.05 was considered statistically significant.
Results
GPx-1, -2, -3, and -4 are expressed in different oviductal cell types
We localized the cellular mRNA expression of the GPx-1, -2, -3, and -4 along the bovine oviduct using in situ hybridization. Our results revealed a cell-specific distribution between cell types for each of the GPx mRNAs in the different oviduct segments. The cellular distribution of GPx-1, -2, -3, and -4 in isthmus, ishtmic-ampullary junction and ampulla is consistent throughout the estrous cycle (data not shown). The classic or cytosolic glutathione peroxidase, GPx-1, is strongly expressed in all oviduct segments and all oviductal cell types (Fig. 1, A–C). Relative quantification revealed that GPx-1 was mostly expressed in the lamina propria and smooth muscle cells of the isthmic-ampullary junction and the ampulla sections (Table 2). Furthermore, the patterns of expression for the gastrointestinal GPx-2 and the extracellular GPx-3 also differed in the three oviduct segments (Fig. 1, D–I; Table 2). Indeed, GPx-2 was found in the oviductal epithelium and the lamina propria, the connective tissue containing blood vessels and free glands blending with the submucosa, of all oviduct sections. Furthermore, positive staining for GPx-2 was also seen in the smooth muscle layer of the ampulla (Fig. 1F; Table 2). In the isthmus and the ishtmic-ampullary junction, GPx-3 expression is mainly detected within the lamina propria (Fig. 1, G and H). In the ampulla, GPx-3 expression is also observed in the single layer epithelium and in the thin smooth muscle layer (Fig. 1I; Table 2). In comparison to GPx-1 to -3, GPx-4 has the most exclusive expression pattern, being restricted to the epithelium in the three oviduct segments (Fig. 1, J–L). Indeed, the intense GPx-4 signal found in the epithelium along the oviduct contrast with the very weak blue staining detected in the circular smooth muscle layer (Table 2).
FIG. 1. In situ localization of GPx transcripts along the bovine oviduct. Transversal cryosections of isthmus, ishtmic-ampullary junction, and ampulla segments were probed with DIG-labeled antisense GPx-1 to -4 RNA probes. GPx mRNAs were detected by immunostaining using alkaline phosphatase-labeled anti-DIG, which appear as a blue staining after incubation with NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) substrate. GPx-1 was ubiquitously expressed in the three sections of the oviduct (A–C). GPx-2 was strongly expressed in the epithelium and was also detected in the lamina propria (D–F) and in the smooth muscle layer of the ampulla (F). GPx-3 expression was mainly located in the lamina propria of the isthmus (G), in the lamina propria, the epithelium and the smooth muscle layer in other segments (H and I). GPx-4 mRNA was primarily detected in oviductal epithelium (J–L). Sense probes were used as negative control (M–O, representative controls). Sections were counterstained with neutral red to visualize cell nuclei. Sense probes were used as negative controls and showed that a nonspecific signal is observed only in the serosa region, and thus was not considered in our interpretation of the results. EP, Epithelial cells; L, oviduct lumen; LP, lamina propria; SE, serosa; SM, smooth muscle cells. Final magnification, x100 and x400.
TABLE 2. Relative quantification of GPx-1 to -4 in the oviduct following in situ hybridization studies (see Fig. 1).
Modulation of GPx-4 expression in the oviduct throughout the estrous cycle
The overall oviductal expression pattern of GPx-4 was determined for the four different stages of the estrous cycle by quantitative RT-PCR. Importantly, GPx-4 expression was highest during the follicular (d 18–20) and postovulatory (d 0–3) stages of the estrous cycle in comparison to the mid-luteal (d 10–12) and the late luteal (d 15–17) stages (Fig. 2, A). To further investigate the regional expression of GPx-4 along the oviduct, three equidistant sections of the oviduct were examined at the same four stages of the cycle (Fig. 2, B–D). Analysis revealed that GPx-4 mRNA expression throughout the estrous cycle varies between the different oviduct sections (Fig. 2, B–D). GPx-4 expression in the isthmus follows the trend observed for the whole oviduct with the highest levels of expression during the d 18–20 and 0–3 (Fig. 2B). In the ishtmic-ampullary junction, GPx-4 expression increases during the follicular and postovulatory stages, but no significant variation is observed between the postovulatory and the late-luteal stages (Fig. 2C). In the ampulla, homogenous GPx-4 expression is present during the postovulatory, the mid-luteal, and the late-luteal stages (Fig. 2D).
FIG. 2. GPx-4 mRNA expression in the bovine oviduct throughout the estrous cycle. The mRNA levels were measured by real-time quantitative PCR. GPx-4 mRNA expression was quantified in the whole oviduct (A), in the isthmus (B), the ishtmic-ampullary junction (C), and the ampulla (D) segments of the oviduct. Expression of GPx-4 at the postovulatory (d 0–3), the mid-luteal (d 10–12), the late-luteal (d 15–17) and follicular (d 18–20) stages of the estrous cycle was compared for each section. The 18S rRNA was used as an internal standard and results are expressed as a ratio of GPx-4/18S. Data are means ± SEM of five animals. Means with different designations (*, ) are significantly (P < 0.05) different from each other (Duncan’s test).
Expression of GPx-4 in the ipsilateral and contralateral oviducts during the estrous cycle
To determine whether the expression of GPx-4 was affected by the proximity of the cycling ovary, oviducts ipsilateral and contralateral to the corpus luteum were analyzed (Fig. 3, A–C). We examined ovaries to select the oviducts from cows characterized only by a change of ovulation side between two consecutive estrous cycles. We observed that in the isthmus and the ishtmic-ampullary junction, GPx-4 expression was highest in the oviduct proximal to the dominant follicle during the follicular phase (Fig. 3, A and B). During the postovulary stage, the GPx-4 mRNA expression is up-regulated in the oviduct ipsilateral to the ovulation side in these sections. Interestingly, GPx-4 expression was not modulated between the ipsilateral and the contralateral oviducts in the ampulla area during the follicular and the postovulatory stages of the estrous cycle (Fig. 3C).
FIG. 3. Differential GPx-4 mRNA expression between the ipsilateral and contralateral oviducts. The mRNA levels were measured by real-time quantitative PCR. The 18S rRNA was used as an internal standard and results are expressed as a ratio of GPx-4/18S. GPx-4 mRNA levels in the ipsilateral () and contralateral () oviducts at the postovulatory (d 0–3) and follicular (d 18–20) stages of the estrous cycle were quantified in the isthmus (A), the ishtmic-ampullary junction (B) and the ampulla (C). Data are means ± SEM of eight animals. Means with designations (*) are significantly different at P < 0.05 (Duncan’s test).
GPx-4 enzymatic activity in the ipsilateral and contralateral oviducts
To determine whether GPx-4 expression patterns paralleled function, its enzymatic activity was measured in whole oviducts at same three stages of the estrous cycle. Our results indicated that the GPx-4 activity was modulated throughout the estrous cycle because significant changes were observed between the three stages of the cycle (Fig. 4A). The highest GPx-4 activity was observed during the postovulatory stage, and the lowest activity was measured during the mid-luteal phase. Detailed analysis of GPx-4 activity in isthmus sections from the ipsilateral and contralateral oviducts revealed that the specific activity was higher in the ipsilateral isthmus during the postovulatory stage, and in the contralateral isthmus during the follicular stage (Fig. 4B). This was consistent with mRNA expression (Fig. 3A). GPx-4 activity was similar in isthmus from both sides at the mid-luteal stage (d 10–12), and no significant variation in GPx-4 activity was also observed between the ipsilateral and the contralateral ampulla sections at all stages of the estrous cycle (Fig. 4C). The results for the enzymatic activities are therefore in accordance with mRNA expression. Interestingly, GPx-4 expression and activity are up-regulated during the estrogen-high phases of the estrous cycle (Figs. 2A and Fig. 4A).
FIG. 4. Analysis of GPx-4-specific activity in the bovine oviduct throughout the estrous cycle. Enzymatic activity was measured by a coupled enzymatic assay at 340 nm. The phosphatidylcholine hydroperoxide was used as substrate. One unit of GPx-4 activity is defined as the amount of protein required to oxidize 1 μM of NADPH/min. A, GPx-4 activity was measured in the whole oviduct at the post ovulatory (d 0–3), mid-luteal (d 10–12) and follicular (d 18–20) stages of the estrous cycle. Data are means ± SEM of eight animals. B and C, Representation of GPx-4 activity in the isthmus and the ampulla segments from ipsilateral () and contralateral () oviducts at the three stages of the estrous cycle. Data are means ± SEM of three animals. Means with different designations (*, ) are significantly (P < 0.05) different from each other (Duncan’s test).
Effects of 17?-estradiol on GPx expression in the oviducts in vivo
Because GPx-4 expression appeared to be regulated during the estrous cycle and influenced by the proximity of the dominant follicle, heifers were treated with intrauterine infusions of 17?-estradiol to test the hypothesis that estradiol up-regulates GPx-4 expression in the bovine oviduct. The mRNA expression of the oviductin, an oviduct-specific glycoprotein known to be regulated by estradiol, was measured by quantitative RT-PCR to ensure estrogen exposure was effective. Estrogen treatment increased expression of oviductin 3-fold in the isthmus and the ampulla (Fig. 5A). Confirming estrous cycle modulated expression of GPx-4, expression was 2-fold higher in the oviducts of cows treated with uterine infusions of 17?-estradiol (Fig. 5E). The effects of the treatment on the transcription of GPx-1 to -3 also were investigated, but no significant variations of the mRNA levels were observed (Fig. 5, B–D).
FIG. 5. Modulation of oviductal GPx-4 expression by 17?-estradiol treatment. The mRNA levels were measured by real-time quantitative PCR (LightCycler). The 18S rRNA was used as an internal standard and results are expressed as a ratio of GPx-4/18S. Cows received either uterine infusion of saline control ( ) or 17?-estradiol () at d 14 of the estrous cycle. The mRNA levels of each gene were quantified in the isthmus and ampulla segments of the oviducts from both groups. A, Oviductin. B, GPx-1. C, GPx-2. D, GPx-3. E, GPx-4. Data are means ± SEM of three animals. Means with designations (*) are significantly different at P < 0.05 (Duncan’s test).
Discussion
We have previously reported that the classical intracellular GPx-1, the gastrointestinal GPx-2, and the extracellular GPx-3 were expressed differentially along the oviduct, in addition to being modulated during the estrous cycle (22). Here, we performed the cellular localization of these genes within the oviduct and found unique expression patterns for each GPx. The classical GPx-1 was ubiquitously expressed in all oviductal cell types. The results for GPx-1 were expected because this gene is known to be expressed in almost all cells of mammalian tissues (24). GPx-1 is involved in the detoxification of various intracellular free hydroperoxides. We also demonstrated that GPx-2 was strongly expressed in the epithelial cells of the oviduct. Similarly, GPx-2 was detected in the mucosal epithelium of the gastrointestinal tract (57). In the intestine, GPx-2 is believed to be involved in the neutralization of ingested LOOHs and in the suppression of inflammation (57, 58). The localization of GPx-3 expression in the lamina propria and in luminal epithelial cells of the ampulla is consistent with a role in regulation of extracellular hydroperoxide levels in the oviductal connective tissues and lumen. It is known that extracellular LOOHs can negatively affect the integrity of sperm and even prevent gamete fusion in vitro (7, 18, 25). GPx-3 is expressed at significant levels in the male genital tract, and the protein is found in the lumen of the caput epididymis (59). Because a sperm reservoir in the initial segment of the oviduct has been uncovered in several mammalian species, it is reasonable to believe that the presence of GPx-3 in this specific site of the oviduct must be important to prevent deleterious effects of luminal LOOHs on male gamete integrity (5). Thus, the cellular localization as well as the modulation of the expression of GPx-1, -2, and -3 suggests that these enzymes may be important regulators of free hydroperoxides levels in the oviduct.
This is the first report of GPx-4 localization in the oviduct. Strikingly, it was mainly expressed in the oviductal epithelium placing it close to gametes found in the lumen or bound to membrane during the follicular and the postovulatory stages of the estrous cycle. This discovery is of high importance because GPx-4 is the only GPx family member with the ability to reduce lipid hydroperoxides bound to cell membranes (24). Lipid hydroperoxides have high potential to affect key oviduct functions such as gamete fusion and/or prostaglandin synthesis (19, 32), so, potential mitigation of their effects by GPx-4 could be essential to fertility.
We observed a direct relationship between the mRNA expression and the specific enzymatic activity for GPx-4. Thus, the mRNA and enzymatic activity levels follow exactly the same trend throughout the estrous cycle as well as in between the ipsilateral and contralateral oviducts. Our data clearly revealed that GPx-4 expression in the oviduct is affected by the proximity of the cycling ovary. Indeed, we reported that GPx-4 is mostly expressed in the oviduct contralateral to the regressing corpus luteum during the follicular phase and in the oviduct ipsilateral to the ovulation site during the postovulatory stage. This latter observation is in concordance with the results of a recent transcriptomics study performed on bovine oviductal epithelium. Indeed, this study showed that GPx-4 was mostly expressed in the ipsilateral oviductal epithelium at d 3.5 after standing heat by using subtractive hybridization and cDNA array hybridization techniques (60). Although this experiment was performed only at the postovulatory stage, it was proposed that the expression of GPx-4 in the ipsilateral oviduct may be up-regulated in response to the presence of the nonfertilized oocyte or due to signals from the active ovary. Our current observations at three stages of the estrous cycle demonstrate that the expression of GPx-4 is up-regulated in the contralateral oviduct during the follicular phase, thus before the release of the oocyte from the dominant follicle. This strongly suggests that GPx-4 expression in the oviductal epithelium is regulated by a signal from the active ovary rather than in response to the presence of the oocyte.
The mammalian oviduct is known to be under the influence of local steroids, especially the estrogens (61, 62). The estrogens are known to induce the transcription of specific genes in the mammalian oviduct (1, 63, 64, 65). Estrogens regulate the proliferation and differentiation of oviductal epithelium and are probably important for oviductal contraction and secretion (65, 66, 67, 68). The highest concentration of estradiol in the bovine oviduct occurs at the end of the estrous cycle during the follicular phase. Furthermore, estradiol levels are higher in the oviduct located near the dominant follicle, suggesting that the estradiol is locally delivered to the oviduct by this follicle (66). Consistent with estrogen regulation, our analysis clearly demonstrated that the higher GPx-4 expression in the oviduct occurs during the maturation of the dominant follicle and the postovulatory period, whereas GPx-4 expression is at its lowest in the ipsilateral and contralateral oviducts during the luteal phase (Fig. 5).
This prompted us to analyze the in vivo effects of the estradiol on the GPx-4 mRNA expression in the bovine oviduct. Here, we provide the first in vivo demonstration that GPx-4 is up-regulated by estradiol in the female reproductive tract. We found that GPx-4 is positively regulated by estradiol in both the isthmic and ampullary regions of the bovine oviduct. Recently, it was reported that estradiol could also increase GPx-4 mRNA expression in male reproductive organs in the rat (69). The effects of estrogens on antioxidant enzymes have been investigated in others tissues. Recently, it was shown that the estradiol induces manganese superoxide dismutases and extracellular superoxide dismutase in vascular smooth muscle cells (70). Moreover, further studies have revealed that total GPx activity was stimulated by the estrogens in human endometrium and in the rat liver (71, 72). Taken together, these observations suggest that estrogens may regulate the oviductal antioxidative pathway by positively affecting antioxidant defense mechanism. We showed that the estradiol induced GPx-4 expression, but the expression of the GPx-1, -2, and -3 remained unchanged. GPx-4 is considered to be the main line of enzymatic defense against oxidative biomembrane destruction and is thought to be of outstanding importance (40, 73). We propose that the stimulation of GPx-4 by estrogens enhance the protection of the oviductal epithelial cell membrane during the follicular and postovulatory stages.
The cellular mechanisms that mediate the actions of estrogen on GPx-4 expression will need further investigation, but several estrogen-responsive elements were located within the 5'-untranslated region of porcine GPx-4 (74). The ERs and ? are expressed in the bovine oviductal cells at all stages of the estrous cycle (75). Interestingly, ER is up-regulated in the isthmus and the isthmic-ampullary junction during the follicular stage, but expression in the ampulla remains unchanged throughout the estrous cycle. The comparison of GPx-4 mRNA expression between the luteal and the periovulatory periods of the estrous cycle also revealed that modulation of GPx-4 is higher in the isthmus than in the ampulla. Furthermore, Gpx-4 is not differentially expressed between the ipsilateral and contralateral oviducts at the ampulla level during the follicular and the postovulatory stages. These results suggest that during the estrous cycle the effects of the estradiol on GPx-4 expression mainly occur within the isthmus and the ishtmic-ampullary junction. The periovulatory period is characterized by an increase in the synthesis and secretion of lipids and others molecules by the bovine oviduct epithelial (50, 76). Thus, the high levels of GPx-4 activity throughout this phase may prevent deleterious effects of lipid peroxidation on oviductal epithelial cell and could have important consequences to these cells physiological functions.
In summary, we have reported that GPx-4 expression and enzymatic activity in the bovine oviduct are modulated in the estrous cycle in addition to being differentially regulated between the ipsilateral and contralateral oviducts. This is the first time that the expression of a specific gene is shown to be regulated by the proximity of the dominant follicle in mammalian oviduct in vivo. Furthermore, despite that it is becoming clear that GPx-4 plays multiples roles in cellular functions, almost nothing is known about the mechanisms of control of GPx-4 expression by extracellular signals. Here, we showed that GPx-4 expression was up-regulated by estradiol in the oviduct and provided the first in vivo demonstration of GPx-4 regulation by endocrine hormones in mammalian cells. Moreover, to support this result, we have established a relationship between GPx-4 expression pattern and estradiol physiological distribution in the oviducts throughout the estrous cycle. The analysis of the role of GPx-4 in oviductal function as well as the characterization of the mechanisms of GPx-4 regulation by estrogen will be important subjects for future investigations.
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