Molecular Human Reproduction, Vol. 5, No. 10, 914-919,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of ovarian function |
Immunolocalization of glutaredoxin in the human corpus luteum
1 Departments of Pathology and 2 Cell Biology, Physiology and Immunology, Faculty of Medicine and 3 Department of Biochemistry and Molecular Biology, Veterinary Faculty, University of Córdoba, 14071 Córdoba, Spain
Abstract
Glutaredoxin (Grx) is a small protein with oxidoreductase activity which is involved in the cellular defence against oxidative stress. Corpus luteum (CL) regression has been related to the generation of reactive oxygen species (ROS). We have studied the presence of glutaredoxin in the human ovary during the ovulatory cycle using polyclonal antibodies developed against recombinant human Grx. Immunostaining was only detected between days 15 and 23 of the cycle and was localized exclusively in the corpus luteum. Grx-positive cells corresponded to granulosa-derived luteal cells (GLC) whereas the remaining luteal cell types were not immunostained. In general, Grx immunoreactivity was parallel to the functional activity of the CL. Most GLC were immunostained on days 15–16 of the cycle, whereas on days 17–19 immunoreaction was found mainly at the inner and outer aspects of the granulosa lutein layer (GLL). After this stage only isolated GLC showed Grx immunoreactivity and no reaction was found from day 23 of the cycle onward. In two CL of pregnancy that were also studied, isolated GLC showed Grx immunoreactivity. Loss of Grx immunoreactivity was coincident with the appearance of morphological signs of structural luteolysis, such as shrinkage of the GLL and the presence of apoptotic cells. These data suggest that Grx, as a cellular antioxidant, plays an important role in the mechanisms of human CL development.
corpus luteum regression/glutaredoxin/luteinization/oxidative stress/redox regulation
Introduction
The human corpus luteum (CL) is a transient endocrine organ that is needed to provide the high progesterone concentrations required to prepare the uterus for implantation and to maintain pregnancy. Nevertheless, in non-fertile menstrual cycles, the CL has a very limited life span. By about day 24 of the standard cycle progesterone secretion sharply declines, which is known as functional regression, and this is followed at the end of the luteal phase by structural luteolysis (Rothchild, 1981
; Farin et al., 1988). The factors controlling the CL life span, as well as the actual mechanisms of luteal cell death, are not fully understood. Two hormones, luteinizing hormone (LH)/chorionic gonadotrophin (CG), as a luteotrophic factor, and prostaglandin F2
(PGF2), as a luteolytic factor, are the main endocrine regulators of the CL in many species (Rothchild, 1981; Farin et al., 1988). Recently, the existence of apoptotic cells has been demonstrated in the CL of several species and apoptosis has been proposed as a possible mechanism of CL regression (Juengel et al., 1993; Tilly, 1996; Young et al., 1997) although alternative cell death pathways have also been reported during luteolysis in primate species (Young et al., 1997).
Several lines of evidence have suggested that changes in the redox state of luteal cells may be one of the primary determinants of the CL life span (Riley and Behrman, 1991; Carlson et al., 1993; Rueda et al., 1997a). Major antioxidants such as ascorbic acid (vitamin C), -tocopherol (vitamin E) carotenoids and glutathione are present in high concentrations in the ovary (Aten et al., 1992) as well as antioxidative enzymes (Rueda et al., 1995). Several studies have pointed out the existence of a link between luteotrophic/luteolytic factors and the production/elimination of reactive oxygen species (ROS) in the CL (Tilly, 1996; Rueda et al., 1997b). In this sense, treatment with the luteotrophin human chorionic gonadotrophin (HCG) increases the expression of antioxidant enzymes in the rat CL (Laloraya et al., 1988) whereas treatment with the luteolytic factor prostaglandin F2 generates free radical species in luteal cells (Sawada and Carlson, 1991; Aten et al., 1998) and depletes ascorbic acid reserves (Aten et al., 1992). Similarly, oxidative stress has been proposed as a determinant of granulosa cell demise in atretic follicles (Tilly and Tilly, 1995) in the rat. Previous studies have reported that the antioxidant reserve of the ovary is endocrine-regulated (Aten et al., 1992). However, information regarding the presence of antioxidant systems in the human CL is scarce.
Glutaredoxin (Grx) is a small protein with thiol-disulphide oxidoreductase activity (Holmgren, 1989; Wells et al., 1993) that catalyses the transfer of electrons from reduced glutathione to several disulphides. The broad spectrum of cell functions in which thiol-disulphide exchange reactions are involved is indicative of the potential roles of oxidoreductases in cell physiology and in pathological conditions. Under oxidative stress, some proteins undergo reversible S-glutathionylation, that is, the formation of a mixed disulphide with glutathione at a key cysteine residue, a process that drastically alters the function of the protein (Chai et al., 1994; Schuppe-Koistinen et al., 1994). It has recently been observed that Grx efficiently catalyses the deglutathionylation process, so contributing to the recovery of protein functionality (Jung and Thomas, 1996). Recovery of the DNA-binding activity by the proliferation-related nuclear factor I (NFI) (Bandyopadhyay et al., 1998) and regulation of protease activity in HIV-1-infected cells (Davis et al., 1997) are two relevant examples. Previous studies have shown that Grx is highly expressed in immunocompetent cells and in epithelial cells of secretory tissues, as well as in the oocytes of growing follicles in the calf (Padilla et al., 1992; Rozell et al., 1993). In the human ovary, the expression of thioredoxin, a related protein with a similar oxidoreductase activity, has been reported in pre-ovulatory follicles and the steroidogenic cells in corpora lutea (Iwai et al., 1992).
In this context, we have studied the expression of Grx in the human CL during luteinization, maturity and regression, as well as in the CL of pregnancy, using a polyclonal antibody against recombinant human Grx.
Materials and methods
Tissue samples
Tissue samples were obtained from the archives of the Department of Pathology from hysterectomized–ovariectomized menstruating women with no clinical history of endocrine pathology. The stage of the cycle was determined by considering menstrual history, dating of the endometrium (Dallenbach-Hellveg, 1971) and corpus luteum (Corner, 1956). Only samples in which these parameters were concordant were considered. The day of ovulation (day 14 of the standard cycle) was considered as day 0. At least three samples per day of the luteal phase (days 14–27 of the standard cycle, days 0–13 of the CL), and some samples in the follicular phase (days 1–13 of the cycle, days 28–40 of the CL) were studied. Corpora lutea were classified as young (1–5 days of age; days 15–19 of the cycle), mature (6–10 days of age; days 20–24 of the cycle), old (11–13 days of age; days 25–27 of the cycle) and regressing (14 or more days of age; follicular phase). In addition, two CL of pregnancy during the first–second month were also studied.
Generation and characterization of glutaredoxin antibodies
Recombinant human Grx was over-expressed in E.coli and purified as described previously (Padilla et al., 1996). The pure protein was used to immunize New Zealand albino rabbits according to a published protocol (Harboe and Ingild, 1983). Preimmune serum was withdrawn before any immunization. The titre of the antisera was determined by inhibition of Grx in the HED reductase assay (Holmgren, 1989); those antisera with high titre were pooled and the IgG fraction was purified by affinity chromatography on a Protein A–Sepharose column.
For the characterization of the antibodies, human placenta stored at –80°C after delivery was obtained from the Hospital Reina Sofía of Córdoba. A crude extract was prepared by homogenization in 4 volumes of TE buffer (50 mmol/l Tris–HCl, pH 7.6, 1 mmol/l EDTA, 0.2 mmol/l dithiothreitol to which 1 mmol/l phenylmethylsulphonylfluoride had been added). Homogenization was achieved with an Ultra-Turrax homogenizer and the supernatant obtained after centrifugation at 27 000 g was used for further study. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis was performed on homogeneous 12% acrylamide gels followed by Coomassie staining or electrophoretic semi-dry transfer to polyvinylidene difluoride membranes. The membranes were processed by Towbin's method (Towbin et al., 1979). The primary antibodies were used at 1:100 dilution and developed with alkaline phosphatase-conjugated goat anti-rabbit IgG and nitroblue tetrazolium/bromo-chloro-indolyl phosphate as chromogen. Protein concentration was determined spectrophotometrically (Bradford, 1976).
Immunohistochemistry
Grx immunostaining was performed on 5 µm thick paraffin sections, which were placed on poly-L-lysine coated slides. After dewaxing in xylene and rehydration in ethanol, endogenous peroxidase was inhibited by incubation in 2% hydrogen peroxide in methanol for 30 min to inhibit endogenous peroxidase. After washing in PBS, sections were blocked with normal rabbit serum and incubated overnight with the primary antibody (anti-human Grx 1:40). The sections were then processed according to the avidin–biotin–peroxidase complex (ABC) following previously described procedures (Gaytán et al., 1997). Sections were counterstained with haematoxylin. Negative controls in which the first antibody was substituted by preimmune serum or PBS were run routinely.
Results
Affinity-purified antibodies equally inhibited the activity of a pure preparation of human recombinant Grx and that of a crude extract from human placenta (Figure 1). Immunoblotting analysis of a crude extract from this human tissue showed a unique intense band, and background noise produced by non-specific binding was very weak even under the `heavy' conditions employed (Figure 2B). This positive protein was barely detected as a faint band with the Coomassie stain in the same position as the reference Grx monomer (Figure 2A).
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On days 14–15, newly formed corpora lutea showed considerable variation. Granulosa–lutein cells (GLC) showed scanty cytoplasm and were slightly luteinized. Most GLC showed cytoplasmic immunostaining for Grx (Figure 3A,B
), although in some samples from day 14 immunostained cells were absent. On days 16–19, GLC showed increasing luteinization, evidenced by the increase in the size of the nucleus and cytoplasm. Strong Grx immunostaining was observed, particularly at the inner and outer aspects of the granulosa–lutein layer (GLL; Figure 3C–D), whereas in the centre of the GLL immunostaining was faint or absent. Grx-immunostained cells corresponded to parenchymal luteal cells, whereas stromal cells were negative. From day 20 to day 24, the number of Grx-immunostained cells decreased. They were found as isolated cells with intense cytoplasmic staining that was localized at the peripheral cytoplasm (Figure 3E,F). They were also located at the inner and outer aspects of the GLL. From day 25 onward, immunostained cells were absent. At this time, morphological signs of CL regression were evident. There was a general shrinkage of the GLL, a decrease in the size of the nucleus and cytoplasm, as well as vacuolization of some GLC. In the CL of pregnancy, isolated GLC were immunostained. Similar to the mature CL, immunostained cells were mostly found at the inner and outer aspects of the GLL, although isolated Grx-positive cells were also found in the centre of the GLL (Figure 3H,I).
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We have previously reported the presence and numbers of macrophages, and proliferating and apoptotic luteal cells during the menstrual cycle (Gaytán et al., 1998
). A schematic representation of the changes in these parameters during the CL life span is presented in Figure 4, for comparison with Grx immunoreactivity.
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Discussion
This is the first study providing evidence on the presence of Grx in the human CL. The specificity of the antibodies used was checked with a crude extract from human origin and was clearly demonstrated by titration of the Grx activity and by Western blotting as shown in Figures 1 and 2
.
Grx immunostaining in the human ovary was limited to the CL. Other structures such as oocytes, ovarian follicles and stroma were negative (see Figure 5), whereas in the bovine ovary strong immunostaining was found in the oocytes of growing follicles (Rozell et al., 1993), although corpora lutea were not investigated. From histological and biochemical studies, it has been established that Grx is expressed in most mammalian cell types, although differentially (Gan and Wells, 1988; Rozell et al., 1993; Martínez-Galisteo et al., 1995). Consequently, we cannot rule out the presence of Grx in other structures of the human ovary at levels that fall below the sensitivity threshold of the immunohistochemical assay under our conditions. This would also explain the lack of staining of red blood cells and macrophages which are known to contain Grx. Hence, our results indicate that the concentration of Grx was considerably higher in the CL than in other ovarian structures and that this protein is highly expressed at a specific stage of the CL life span.
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The peculiar morphological features of large GLC (large cells with a polygonal cytoplasm and round nuclei), as well as their segregation from small theca–lutein cells in women, allowed us to identify Grx-immunostained cells as GLC. No other luteal cell types (i.e. fibroblasts, endothelial cells, pericytes or macrophages) were immunostained. The expression of Grx has been demonstrated by immunohistochemistry in spleen macrophages and in other immunocompetent cells in lymphoid tissues (Rozell et al., 1993
). However, in the present study, CL macrophages were negative. Although double immunostaining for Grx and the macrophage marker CD68 was not performed, the presence of Grx immunostaining in macrophages can be discarded. First, Grx was expressed in most cells of the GLL on days 15–16, when macrophages were still scarce, and second, macrophages showed distinctive morphological features in CD68-immunostained sections (Gaytán et al., 1998).
The human CL undergoes dramatic changes from follicle rupture (on day 14 of the standard cycle) to the end of the luteal phase (on day 27 of the cycle). These changes involve luteinization of the granulosa and theca cells, proliferation of both stromal and parenchymal cell populations during early and mid luteal phases coincident with the release of high amounts of progesterone from parenchymal cells, and regressive changes and a sharp decline in progesterone release at the late luteal phase. Finally, complete structural luteolysis leading to the formation of corpora albicantia takes several cycles. The presence of Grx also changed during the CL life span, being roughly parallel to its functional activity. Interestingly, an inverse relationship between Grx immunostaining and CL regression was found. Morphological signs of CL ageing, such as the decrease in the size and vacuolization of GLC, infiltration of lymphocytes, a decrease in the size and numbers of macrophages in the GLL and the presence of apoptotic cells (Shikone et al., 1996; Gaytán et al., 1998) appeared at the end of the mid-luteal phase (days 23–24) and were evident at the late luteal phase (days 25–27). These changes were coincident with the loss of Grx immunostaining. Though limited by the number of samples analysed, the results obtained with CL of pregnancy suggest that there is a prolonged expression of Grx, which would also be in accordance with the maintained functional activity and rescue from luteolysis of the CL.
The promoter region of the human Grx gene contains an AP-1 cis-regulatory element at ~–180 bp upstream of the transcription start point (Park and Levine, 1997). AP-1 transcription factor is known to be activated, among other stimuli, by cAMP (Abate et al., 1990). Since LH acts on CL cells through the protein kinase A cascade, it is likely that the rise in Grx expression is part of the response of luteal cells to the LH surge that precedes luteinization. The consumption of reducing equivalents by the intense proliferative and steroidogenic activities of these cells would lead them to oxidative stress conditions were it not for the display of increased antioxidant enzymes, of which superoxide dismutase (Laloraya et al., 1988) and Grx (this study) seem to be part. The observed decrease in Grx expression that follows the initial surge would provoke the weakening of antioxidant defences. This in turn, would allow for the increase in ROS which is observed in the CL during the mid-luteal phase (Vega et al., 1994) and paves the way to CL involution (Riley and Behrman, 1991; Carlson et al., 1993). The decline of Grx concentrations in the course of CL development could be triggered by agent(s) acting on negative regulatory elements known to be present further upstream in the Grx gene promoter (Park and Levine, 1997).
The presence of Grx immunoreactivity was, in general, coincident with the proliferative activity of GLC (Gaytán et al., 1998) both in young and in mature CL. However, Grx was not found in other ovarian structures with high proliferative activity such as growing and pre-ovulatory follicles. Therefore, a relationship exists between Grx immunoreactivity and proliferative activity of GLC but only after the onset of luteinization.
In summary, the presence of Grx immunoreactivity in the human CL during the functional period of the luteal phase and perhaps its maintenance during pregnancy strongly suggest a role for this protein in CL regulation. Additional studies on the mechanisms and precise roles of this protein would be of interest.
Acknowledgments
The authors are very grateful to P.Cano for technical assistance. This work was supported by grants CICYT PB94-0451-CO2-02 and DGESEIC PM98-0167.
Notes
4 To whom correspondence should be addressed 
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Submitted on April 1, 1999; accepted on July 8, 1999.
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