Molecular Human Reproduction, Vol. 6, No. 5, 397-403,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum
1 Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, 413 45 Göteborg, Sweden and 2 Department of Reproductive Medicine, University of California at San Diego, La Jolla, CA, USA
Abstract
The aims of the present study were to characterize the expression and cellular localization of isoforms of nitric oxide synthase (NOS) in the human corpus luteum (CL) and to determine the effects of nitric oxide (NO) on CL steroidogenesis. Immunoblotting analyses revealed that endothelial NOS (eNOS) is the most abundant isoform in human CL with highest values during the late luteal phase. Immunoreactive eNOS was localized predominantely in the theca lutein layer, being particularly abundant in endothelial cells, but with positive staining also in some steroidogenic cells. Immunoreactive inducible NOS (iNOS) was also detected, but to lesser degree, and did not display apparent phase-specific changes. The effect of NO on CL steroid synthesis was examined using human chorionic gonadotrophin (HCG)-stimulated dispersed CL cells cultured in vitro. Progesterone production was significantly decreased (P < 0.05) by the NO donor spermine NONOate (10–5 mol/l) in cells of the late, but not mid-, luteal phase. To investigate a potential link between NO and the local prostaglandins (PG), concentrations of PGF2
and PGE2 were measured in culture medium. NO significantly increased (P < 0.05) concentrations of both PGF2 and PGE2 during the late luteal phase. It is concluded that NO may be luteolytic in the human CL of menstruation.
corpus luteum/eNOS/NOS/nitric oxide/prostaglandins
Introduction
In recent years, nitric oxide (NO) has been recognized as a paracrine molecule that evokes cell function via gas diffusion, a truly new concept in cell to cell signalling (Moncada and Higgs, 1993
). NO participates in a variety of general physiological and pathophysiological processes such as endothelial-derived relaxation of smooth muscle cells in precapillary blood vessels (Thom et al., 1985), inflammation (Ialenti et al., 1993), and neurotransmission (Garthwaite, 1991). In the female genital tract, NO is involved in regulating a wide variety of processes, including cervical ripening (Thomson et al., 1997), oviduct motility (Ekerhovd et al., 1997), uterine relaxation (Buhimschi et al., 1995), and ovarian processes such as ovulation (Bonello et al., 1996).
NO is produced by the conversion of L-arginine to L-citrulline by NO synthase (NOS). Two isoforms, endothelial NOS (eNOS) and inducible NOS (iNOS), have been detected in ovarian tissues of the rat and mouse (Van Voorhis et al., 1995; Olson and Jones-Burton, 1996; Jablonka-Shariff and Olson, 1997), whereas the third isoform, neuronal (nNOS), has not yet been detected in the ovary. In rat ovaries, immunoblotting experiments revealed that eNOS is present in larger quantities than iNOS, with both isoforms being distributed predominantly in the ovarian stroma, the follicular theca, and the corpus luteum (CL) (Zackrisson et al., 1996; Jablonka-Shariff and Olson, 1997). In the human, eNOS mRNA is expressed in human luteinized granulosa cells (Van Voorhis et al., 1994) and CL (Vega et al., 1998), indicating that these cells may produce the peptide.
The primary function of the CL is to produce large amounts of progesterone, a function which is accompanied by the development of an extensive vascular system (Gaytán et al., 1999) which becomes maximally dilated (Wiltbank et al., 1990). The dilatation of the vascular bed, which may be NO-mediated, and the presence of eNOS mRNA could suggest a role for NO in luteal processes. Indeed, there is evidence that NO decreases steroidogenesis in rat lutein cells (Olson and Jones-Burton, 1996) and in hyperstimulated luteinized granulosa cells obtained from IVF patients (Van Voorhis et al., 1994). Consistent with this, is the indirect evidence that the NO precursor L-arginine and an unselective pharmacological NOS inhibitor modulate basal steroid synthesis in human CL of the mid-luteal phase (Vega et al., 1998).
The aims of the present study were firstly to examine in more detail the concentrations and localization of NOS isoforms in the human CL throughout the luteal phase with special reference to the mid- and late luteal phases and, secondly, to establish whether NO affects human chorionic gonadotrophin (HCG)-stimulated progesterone synthesis during these phases.
Materials and methods
Experimental subjects
A total of 16 women, undergoing surgery at the Department of Gynecology at Sahlgrenska University Hospital for non-ovarian, benign gynaecological conditions, volunteered for the study. All women gave informed consent and the study was approved by the Ethical Committee at Göteborg University.
Histories of the last three menstrual cycles were obtained and only women with cycle lengths of 26–32 days were included in the study. None of the women had taken any hormonal medication for the last 3 months prior to surgery. At surgery, the corpus luteum (CL) was excised in toto at the beginning of the surgical procedure, leaving the ovary intact. The morphology of the CL was examined to confirm the stage of luteal phase based on the patient's history. The CLs were staged as early (days 0–4 after ovulation, n = 2), mid- (days 5–9 after ovulation, n = 7) and late (days 10–14 after ovulation, n = 6). One CL was excised during the follicular phase of the next menstrual cycle and was characterized as regressing. In all, 11 CL were used for cell culture experiments, 11 for Western blotting, and seven CL for immunohistochemistry.
Hormones, chemicals, and antibodies
Spermine NONOate was purchased from Alexis Corporation (Läufelfingen, Switzerland); HCG (Profasi; Serono, Geneva, Switzerland); phosphate-buffered saline (PBS), minimal essential medium (MEM) with Earle's salts, gentamicin, and fetal calf serum (FCS) from Life Technologies Inc (Paisley, Scotland, UK); collagenase (CLS 2; Worthington Biochemical Corporation, Freehold, NJ, USA); DNAse grade 2 (Boehringer-Mannheim, Mannheim, Germany); monoclonal mouse anti-eNOS antibodies (Transduction Laboratories, Lexington, KY, USA); polyclonal rabbit anti-iNOS antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA); secondary alkaline phosphatase-conjugated goat anti-mouse antibodies and goat anti-rabbit antibodies (Tropix, Bedford, MA, USA); 3-[(3-cholamidopropyl)dimetyl-ammonio]1-propane sulphonate (CHAPS; Boehringer Mannheim).
Tissue handling
The CL tissue used for immunoblot and immunohistochemistry was snap-frozen in liquid nitrogen and kept at –70°C until use. Fresh luteal tissue was immediately put into ice-cold Ca2+ and Mg2+-free PBS and the outer fibrous capsule together with the large blood vessels and the central blood clot were removed from the luteal tissue. The CL tissue was then cut into small pieces.
Dissociation and fractionation of cells
The pieces of CL were digested by collagenase CLS 2 (2.5 mg/ml) and DNAse grade 2 (50 µg/ml) under rotatory motion at 37°C for 60 min. The cell suspension was passed through a mesh cell strainer (pore size 100 µm), washed twice in PBS, and the cell filtrate was pelleted by centrifugation at 200 g for 5 min. Following resuspension in PBS, a portion, referred to as all corpus luteum cells (ACLC), was set aside for cell culture. The remainder was fractionated into five cell-bands by centrifugation for 20 min at 400 g on a discontinuous iso-osmolar Percoll gradient (Pharmacia Upjohn, Uppsala, Sweden) of 63, 54, 45, 27, and 18% layers (densities 1.088, 1.076, 1.041, and 1.029 g/mmol/l respectively). Cells from the interfaces at 18/27% and 27/45% were found to secrete >98% of the total progesterone produced by the ACLC. These cells were used for subsequent culture experiments and referred to as enriched steroidogenic luteal cells (ESLC).
In each of the two bands, >75% of the cells expressed 3ß-hydroxysteroid dehydrogenase (HSD) immunoreactivity as a indicator of steroidogenic capacity (Fridén et al., 1999). Samples from each of the two bands as well as from the cell suspension (ACLC) were counted in a haemocytometer and assessed by the Trypan Blue exclusion method. Viability in all cell preparations was >90%.
Cell culture procedures
The ACLC and ESLC were washed three times in cell culture medium (MEM) supplemented with FCS (10%), L-glutamine (292 mg/mmol/l), and gentamicin (50 µg/mmol/l). Cells (75x103/500 µmol/l) were pipetted into 24-well tissue culture plates (Falcon; Becton Dickinson, Franklin Lakes, NJ, US) and precultured for 24 h at 37°C in 5% CO2 in air to promote attachment. After changing the culture medium, cells were incubated for another 24 h in the presence and/or absence of HCG (100 IU/l) and the NO donating drug spermine NONOate. This substance releases NO spontaneously at a predictable rate once in aqueous solution (Marangos et al., 1991). After 24 h the supernatants were collected and stored at –70°C until analysis.
Assays
Progesterone concentrations in medium were analysed in a time-resolved immunofluorometric assay (Delfia; Wallac Oy, Turku, Finland). Both the intra- and inter-assay coefficients of variation were <9.6%. Prostaglandins (PG) F2 and PGE2 were analysed using enzyme-linked imunosorbent assay (ELISA) kits (R&D Systems, Abingdon, UK). The sensitivity of the PGF2 assay was >4.62 pg/ml and cross-reactivity with PGF1 was 20%, with PGE2 0.2% and all other prostaglandins <1%. The sensitivity of the PGE2 assay was > 36.2 pg/ml and cross-reactivity with PGE1 was 70%, and with PGF2 0.7%. All samples were analysed in duplicate or triplicate.
Immunoblotting
Soluble protein from CL was prepared as described previously (Piontkewitz et al., 1993), with minor modifications. CL were homogenized by a polytron in PE buffer (10 mmol/l potassium phosphate buffer, pH 6.8, and 1 mmol/l EDTA) containing 10 mmol/l CHAPS. The homogenate was sonicated (2x10 s) and then centrifuged for 5 min at 12000 g. Samples (50 µg total protein) were diluted in sodium dodecyl sulphate (SDS) sample buffer and loaded on a one-dimensional SDS polyacrylamide gel (4.5% stacking gel, 6–8% separating gel; Novex, San Diego, CA, USA). Gels were run at 125 V for 2 h, after which the proteins were electroblotted onto a polyvinyldifluoride membrane (PVDF; Amersham, Buckinghamshire, UK) and subsequently incubated at 4°C overnight with antibodies against eNOS (dilution 1:100) and iNOS (dilution 1:100). Immunoreactive proteins were visualized by chemiluminescense using alkaline-conjugated secondary antibodies (goat anti-mouse and goat anti-rabbit antibodies respectively) and CDP-Star (Boehringer-Mannheim) as substrate. The filters were exposed to ECL film (Amersham) at room temperature for 1–2 min and subsequently developed. As positive controls recombinant eNOS, rat liver and aorta (eNOS) and rat spleen (iNOS) were used.
Immunohistochemistry
Frozen sections (6 µm) were placed onto gelatin/chrome aluminium-coated slides and air-dried. Slides were treated with 0.3% (w/v) H2O2 in methanol for 30 min, incubated overnight (4°C) with anti-eNOS antibodies (1:100) or anti-iNOS antibodies (1:100) followed by three washes of PBS. A sensitive detection kit (Vecta Stain ABC kit) with secondary antibodies was used. The slides were counterstained in haematoxylin and mounted in Pertex (Histolab, Göteborg, Sweden). Positive staining was visualized as brownish reaction product. As positive controls, human uterine artery (eNOS) and inflamed human lung tissue (iNOS) were used. Negative controls were incubated without the anti-NOS antibodies or with an unrelated antibody against chromogranin A (generously provided by O.Nilsson, Department of Pathology, Göteborg University). Staining intensity and localization was assessed by two independent observers.
Statistical analysis
All individual observations were in duplicate or triplicate. The inter-individual variations in steroid concentrations were large and the values were therefore log-transformed, resulting in normalization of the material acceptable for analysis of variance, followed by Fisher's Exact Least Squares Differences (LSD). P < 0.05 was considered to be statistically significant. The software used for all analyses was Stat-View 4.5 (Abacus Concepts, Berkeley, CA, USA).
Results
Presence of NOs in the human CL
By means of immunoblotting eNOS protein of the predicted 135 kDa size was detected in all tissue samples examined. The intensity of eNOS protein band appeared greater in samples of the late luteal phase (n = 5) when compared with the earlier (early n = 2, mid- n = 2) and regressing stage (n = 1) (Figure 1). Although numbers were small, the mean OD for late CL was ~50% higher than for CL of mid-luteal phase. In Figures 1 and 2, representative results are presented. Inducible NOS was also demonstrated in all samples at the 130 kDA size, but the concentrations in the CL did not show any clear phase variations (Figure 2).
|
|
Immunohistochemistry revealed clear eNOS staining in all examined CL (n = 7), which were obtained from the luteal phase, whereas cells in the regressing CL were not stained by the eNOS antisera. The distribution of eNOS immunoreactivity was similar in all CL, with a typical intense staining of the endothelial cells of blood vessels (Figure 3a
). In the theca lutein area, more numerous cells of steroidogenic appearance contained eNOS immunoreactivity, while fewer and scattered cells of the granulosa lutein area demonstrated immunostaining. Some cells, which were located predominantly in the theca lutein area, stained positively for iNOS (Figure 3b). These cells were large with a multi-angular cytoplasm and did not display typical steroidogenic appearance. No staining was detected in the negative controls (Figure 3c), while staining was detected in all positive controls (data not shown).
|
Effects of NO-donor on luteal steroidogenesis
Studies regarding effects of NO on luteal steroidogenesis were performed on ACLC and ESLC of mid- and late luteal phase. The concentrations of progesterone in the culture medium from cells cultured under non-stimulated conditions were higher in both ACLC and ESLC obtained from CL of the mid-luteal phase as compared with these cells from CL of late luteal phase (Figure 4
). Treatment with HCG (100 IU/l) caused a general mean increase of basal progesterone concentrations, compared with controls of 2.5-fold for mid-luteal cells and 7–8-fold for late luteal cells.
|
To determine a possible role of NO in regulation of LH/HCG driven luteal steroidogenesis, the effects of an NO donor spermine (NONOate) on HCG-stimulated (100 IU/l) steroidogenesis were examined. When luteal cells were cultured for 24 h with increasing concentrations of NONOate (10–6, 10–5, 10–4 mol/l), there was a dose-dependent decrease in progesterone concentrations (96, 32, and 25% of control values at 10–6, 10–5, 10–4 mol/l respectively). Based on these results the NONOate concentration of 10–5 mol/l was chosen for subsequent experiments. No decrease was noted for vehicle only.
The progesterone concentrations were generally significantly (P < 0.0049) lower in the presence of HCG + NONOate in cell fractions from late luteal phase (mean for ACLC 32 and for ESLC 27 nmol/l respectively) as compared with HCG controls (mean 53 for ACLC and for ESLC 49 nmol/l) of the same luteal age. There was no significant difference between cell fractions of mid-luteal phase cultured in the absence or presence of NONOate (Figure 4
). However, when comparing each fraction separately only HCG-control ESLC compared with treated ESLC of late luteal phase displayed significant difference (P = 0.014). The ACLC fraction of late luteal phase did not reach significant concentrations due to large variation in the material.
Effect of NO-donor on prostaglandin synthesis
The concentrations of PGF2 were significantly (P = 0.024) increased in the presence of NONOate for ACLC cells of late luteal phase (HCG: mean 736 pg/ml; HCG+NO: mean 1081 pg/ml). This difference was not noted in cultures of ESLC cells of late luteal phase (Figure 5a). In contrast, ACLC and ESLC cells of the mid-luteal phase did not exhibit any difference in PGF2 concentrations between cultures with HCG alone or HCG+NONOate. Likewise, concentrations of PGE2 significantly increased in the presence of NONOate (P = 0.0002) in ACLC cells from late luteal phase (HCG: mean 1311 pg/ml; HCG+NO: mean 2677 pg/ml) (Figure 5b). No difference was recorded neither for ESLC with treatment of NONAte of any phase nor for ACLC of mid-luteal phase.
|
Discussion
In the present study we have demonstrated the expression of eNOS and iNOS protein in human luteal tissue. The expression of eNOS seems to be time-dependent with higher concentrations during the late luteal phase. We have also demonstrated that the product of these enzymes, NO, have luteolytic effects in vitro on human luteal cells from the late luteal phase, but not the mid-luteal phase.
We observed a different distribution of eNOS and iNOS in the granulosa and theca lutein areas. The predicted intense staining for eNOS in cells surrounding the blood vessels in the CL was observed (Van Voorhis et al., 1995; Vega et al., 1998). In addition, large numbers of steroidogenic appearing cells displayed eNOS staining. These eNOS-positive cells appeared to be more abundant in the theca lutein layer. The inducible isoform, iNOS, was also found in cells located within the theca lutein area, although fewer cells were immunoreactive to iNOS, compared with eNOS. In accordance with a recent study in the human CL (Vega et al., 1998) the iNOS positive cells did not exhibit a typical steroidogenic phenotype, indicating that a subpopulation of tissue-bound leukocytes in the CL are activated and express iNOS.
The tissue architecture and distribution of various cells in the CL differ between species. Previous studies have mostly been designed to localize NOS in the CL of the rat. The rat CL is rather homogenous in structure compared to the human CL and a clear demarcation between theca lutein and granulosa lutein areas is not easily identified. In the commonly used hyperstimulated rat models, stronger staining for eNOS was seen inside the CL, compared with the surrounding stromal areas (Olson and Jones-Burton, 1996; Jablonka-Shariff and Olson, 1997). Previous observations of a fairly general positive eNOS staining inside the rat CL with streaks of stronger staining in a radiating fashion (Zackrisson et al., 1996) may represent infolding of theca-derived cells. This may correspond to the relatively dominating expression of eNOS to the theca lutein layer of the human CL, as found in the present study. In the human CL there is a higher quantity of precapillary NO-responsive vessels in this region. In luteinized tissue from hypophysectomized DES-treated rats, immunohistochemistry revealed that endothelial cells, and some of the parenchymal luteal cells, express NOS (Olson and Jones-Burton, 1996). This fits with our findings of immunoreactive eNOS in theca lutein cells. It also supports a recently published study (Vega et al., 1998), which shows the presence of eNOS in large luteal-like cells in human CL tissue. Taken together, the results of the present and other studies indicate that a subpopulation of the luteal cells in both the rat and the human CL express NO. These cells can by autocrine/paracrine mechanisms influence most of the cells in the CL.
The immunoblotting experiments suggested that the highest concentrations of eNOS are found during the late luteal phase, indicating a functional role for eNOS during this phase. A similar increase of eNOS protein concentrations during the later stages of the luteal phase was seen in the eCG/HCG rat model, where maximal concentrations were seen during days 9–11 of the induced luteal phase (Van Voorhis et al., 1995; Zackrisson et al., 1996; Jablonka-Shariff and Olson, 1997). However, maximal eNOS mRNA expression, as assessed by quantitative reverse transcription–polymerase chain reaction (RT–PCR), was observed during the early and mid-luteal phases with an ~50% reduction during late luteal phase (Vega et al., 1998). The different time profiles of eNOS protein and mRNA expression can be explained by variations in post-transcriptional processing during the various stages of the luteal phase and also to the methodological difficulties involving quantitative RT–PCR. Interestingly, eNOS was barely detectable in the regressing CL obtained during the next follicular phase, either by immunoblotting or by immunohistochemistry. This suggests that NOS activity, particularly that of eNOS, may be important only during the functional luteal phase. The vascularity of the CL changes throughout its life with maximal capillary networks (Gaytán et al., 1999) during the mid-luteal phase. Since the number of endothelial cells and vasculature in the CL decrease during the late luteal phase (Gaytán et al., 1999) when eNOS expression is elevated, it is possible that a considerable increase in eNOS expression takes place in the CL cells during this period of development. Inducible NOS appeared to be present in lower quantities in all CL studied by immunoblotting and immunohistochemistry techniques. The protein concentrations did not display any marked cycle-dependent changes. These findings are in line with earlier reports of expression of iNOS protein in the external layers of developing rat CL and also to a larger degree throughout the degenerating CL in the rat (Jablonka-Shariff and Olson, 1997). Inducible NOS has also been demonstrated in luteinized ovarian cells in vitro (Olson and Jones-Burton, 1996), confirming other studies in the rat (Zackrisson et al., 1996). The advantage of detection of immunoreactive protein is emphasized by earlier studies being unable to demonstrate iNOS mRNA in the rat CL (Van Voorhis et al., 1995).
There is a relatively constant input of LH stimulus throughout the human luteal phase (McLachlan et al., 1989) and the luteolytic events cannot be explained by LH withdrawal. There seems to be a general luteolytic pathway so that disparate mechanisms leading to luteolysis all eventually result in a rapid loss of LH receptors and 3ß-HSD (Duncan et al., 1998). To evaluate the possible contribution of NO to the luteolytic events, we chose to evaluate the effect of NO on HCG-stimulated luteal cells. Human CL cells from natural cycles were used in order to study tissue as physiologically as possible, in contrast to the more widely used luteinized granulosa cells from IVF cycles. It is now accepted that non-steroidogenic cells make up a major proportion of the cells in the CL and that these fibroblasts, endothelial cells, and immune cells play active roles in regulation of CL steroidogenesis (Stouffer and Brannian, 1993; Brännström and Fridén, 1997). Therefore, two fractions of cells were used in the cell culture studies with the purpose of evaluating a possible paracrine influence of non-steroidogenic cells. In the present study, we used a well characterized NO donor, which releases NO spontaneously without enzymatic intervention (Maragos et al., 1991). The NO donor NONOate significantly decreased HCG-stimulated progesterone formation in luteal cells from late luteal phase in culture, suggesting a possible link between NO and luteolysis. These findings extend earlier indirect observations in the rat in vivo (Shukovski and Tsafrir, 1994) and on human IVF granulosa lutein cells in vitro (Van Voorhis et al., 1994) and in porcine granulosa cells (Masuda et al., 1997) where the presence of unselective NO antagonists have been shown to increase progesterone and oestradiol concentrations (Shukovski and Tsafrir, 1994).
In the present study, we found that concentrations of PGF2 were increased by NO donor in the cell fractions from the late luteal phase containing all luteal cells treated with HCG, but not in the enriched fractions of steroid producing cells. This may indicate that one mechanism for luteolysis via NO is by a paracrine pathway involving PGF2 release from paraluteal cells affecting steroidogenesis. PGF2 has been demonstrated to be the major luteolytic agent in many animal species, acting by inhibiting several key steroidogenic enzymes including steroidogenic acute regulating protein (StAR) activity (Stocco and Clark, 1996) and mRNA expression (Chung et al., 1998) and affecting the cholesterol delivery to the steroidogenic enzyme P450scc (Wiltbank et al., 1993). In the human CL, PGF2 inhibits progesterone synthesis, as demonstrated on luteal cells in culture and after intraluteal injection of PGF2 in vivo (Bennegård et al., 1991). In several tissues, NO has been found to up-regulate cyclo-oxygenase 2 (COX-2), the major enzyme responsible for prostaglandin synthesis (Salvemini et al., 1993). The luteolytic response to PGF2 seems to be altered depending on the stage of the CL with newly formed human CL being non-responsive to PGF2 (Dennefors et al., 1982). Our findings are in accordance with those of a recent study (Motta et al., 1999), in which it was found that NO donors given to rats increased PGF2 synthesis in the ovary and decreased progesterone concentrations in serum in a phase-dependent manner. A positive feedback mechanism between PGF2 and NO to ensure luteal regression in the rat was proposed. Based on our results, this mechanism could also be envisaged in the human.
Another step in this chain of events is the fact that interleukin 1ß (IL-1ß) acts luteolytically concurrent with increasing the expression of PGF2 and PGE2 and NO in the early corpus luteum of the rat (Ellman et al., 1993; Hurwitz et al., 1997). In the former study it was demonstrated that the luteolytical effects of IL-1ß did not require the presence of NO, indicating that the luteolytic effects of these two compounds act independently. In the latter study, it was suggested that NO acts as a obligatory mediator of cytotoxicity by IL-1ß.
On the other hand, PGE2, being the other major product of COX-2, seems to stimulate luteal progesterone synthesis (Dennefors et al., 1982). In general, concentrations of PGE2 in the CL are higher than concentrations of PGF2 (Pathwardhan and Lanthier, 1980), which was confirmed in our study. When NO was added to the culture medium a significant increase of concentrations of PGE2 was noted in the late luteal phase for cells of the ACLC fraction. This is in accordance with the notion of NO as inducer of COX-2 with a larger absolute increase of PGE2 in comparison to PGF2.
In conclusion we have identified eNOS and iNOS protein in human CL, more frequent in the theca lutein area and around blood vessels. We have also demonstrated that NO in vitro decreases progesterone and increases PG synthesis in cells from the late luteal phase and can possibly act as a luteolysin.
Acknowledgments
We thank Professor Greg Erickson, La Jolla, USA, for fruitful scientific discussions in preparation of this manuscript. The authors are grateful to Ann Wallin and Birgitta Weijdegård for excellent technical assistance. This work was supported by the Merchant Hjalmar Svensson Foundation, Göteborg, Sweden; Göteborg Medical Association, Göteborg, Sweden, Sahlgrenskas Fonder, and the Swedish Medical Research Council (to M.B.).
Notes
3 To whom correspondence should be addressed 
References
Bennegård, B., Hahlin, M., Wennberg, E. et al. (1991) Local luteolytic effect of prostaglandin F2 alpha in the human corpus luteum. Fertil. Steril., 56, 1070.[Web of Science][Medline]
Bonello, N., McKie, K., Jasper, M. et al. (1996) Inhibition of nitric oxide: effect on IL-ß enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution. Biol. Reprod., 54, 436–445.[Abstract]
Brännström, M. and Fridén, B.E. (1997) Immune regulation of corpus luteum function. Sem. Reprod. Endocrinol., 15, 363–370.[Medline]
Buhimschi, I., Yallampalli, C., Dong, Y.-L. et al. (1995) Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am. J. Obstet. Gynecol., 172, 1577–1584.[Web of Science][Medline]
Chung, P.H., Sandhoff, T.W. and McLean, M.P. (1998) Hormone and prostaglandin F2 alpha regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in human corpora lutea. Endocrine, 8, 153–160.[Web of Science][Medline]
Dennefors, B., Sjögren, A. and Hamberger, L. (1982) Progesterone and cyclic adenosine 3',5'-monophosphate formation by isolated human corpora lutea of different ages: influence of human chorionic gonadotrophin and prostaglandins. J. Clin. Endocrinol. Metab., 55, 102.
Duncan, W.C., Illingworth, P.J., Young, F.M. et al. (1998) Induced luteolysis in the primate: rapid loss of luteinizing hormone receptors. Hum. Reprod., 13, 2532–2540.
Ekerhovd, E., Brännström, M., Alexandersson, M. et al. (1997) Evidence for nitric oxide mediation of contractile activity in isolated strips of the human Fallopian tube. Hum. Reprod., 12, 301–305.
Ellman, C., Corbett, J.A., Misko, T.P. et al. (1993) Nitric oxide mediates interleukin-1-induced cellular cytotoxicity in the rat ovary. A potential role for nitric oxide in the ovulatory process. J. Clin. Invest., 92, 3953–3056.
Fridén, B.E., Hagström, H.-G., Lindblom, B. et al. (1999) Cell characteristics and function of two subpopulations of human luteal cells during prolonged culture. Mol. Hum. Reprod., 5, 714–719.
Garthwaite. (1991) Glutamate, nitric oxide and cell-cell signaling in the nervous system. Trends Neurol Sci, 14,60–67.[Web of Science][Medline]
Gaytán, F., Morales, C., Garcia-Pardo, L. et al. (1999) A quantitative study of changes in the human corpus luteum microvasculature during the menstrual cycle. Biol. Reprod., 60, 914–919.
Hurwitz, A., Finchi-Yeheskel, Z., Yagel, S. et al. (1997) Interleukin-1ß inhibits progesterone accumulation in rat corpora luteal cell cultures in a mechanism dissociated from its effects on nitric oxide and prostaglandin E accumulation. Mol. Hum. Endocrinol., 133, 41–48.
Ialenti, A., Ianaro, A., Moncada, S. et al. (1993) Modulation of acute inflammation by endogenous nitric oxide. Eur. J. Pharmacol., 211, 177–182.
Jablonka-Shariff, A. and Olson, L.M. (1997) Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology, 138, 460–468.
Maragos, C.M., Morley, D., Wink, D.A. et al. (1991) Complexes of NO with neutrophiles as agents for controlled biological release of nitric oxide. Vasorelaxant effects. J. Med. Chem., 34, 3242–3247.[Web of Science][Medline]
Masuda, M., Kubota, T., Karnada, S. et al. (1997) Nitric oxide inhibits steroidogenesis in cultured porcine granulosa cells. Mol. Hum. Reprod., 3, 285–291.
McLachlan, R.I., Cohen, N.L., Vale, W.W. et al. (1989) The importance of LH in the control of inhibin and progesterone secretion by the human corpus luteum. J. Clin. Endocrinol. Metabol., 68, 1078.
Moncada, S. and Higgs, A. (1993) The L-arginine – nitric oxide pathway. N. Engl J. Med., 329, 2002–2012.
Motta, A.B., Esterez, A. and de Gimeno, M.F. (1999) The involvement of nitric oxide in corpus luteum regression in the rat: feedback mechanism between prostaglandin F2 and nitric oxide. Mol. Hum. Reprod., 5, 1011–1016.
Olson, L.M. and Jones-Burton, C.M. (1996) Nitric oxide decreases estradiol synthesis of rat luteinized ovarian cells: possible role for nitric oxide in functional luteal regression. Endocrinology, 137, 3531–3539.[Abstract]
Pathwardhan, V.V. and Lanthier, A. (1980) Concentration of prostaglandins PGE2 and PGF2 alpha, estrone, estradiol and progesterone in human corpora lutea. Prostaglandins, 20, 963.[Web of Science][Medline]
Piontkewitz, Y., Enerbäck, S. and Hedin, L. (1993) Expression and hormonal regulation of the CAAT enhancer binding protein during differentiation of rat follicle. Endocrinology, 133, 2327–2333.
Salvemini, D., Misko, T.P., Masferrer, J.L. et al. (1993) Nitric oxide activates cyclooxygenase enzymes. Proc. Natl Acad. Sci. USA, 90, 7240–7244.
Shukovski, L. and Tsafriri, A. (1994) The involvement of nitric oxide in the ovulatory process of the rat. Endocrinology, 135, 2287–2290.[Abstract]
Stocco, D.M. and Clark, B.J. (1996) Regulation of the acute production of steroids in steroidogenic cells. Endocrine Rev., 17, 221–244.
Stouffer, R.L. and Brannian, J.D. (1993) The function and regulation of cell populations composing the corpus luteum of the ovarian cycle. In Adashi, E.Y. and Leung, P.C.K. (eds), The Ovary. Raven Press, New York, USA, pp. 245–259.
Thom, S.A., Hughes, A.D., Martin, G.N. et al. (1985) The release of the endothelium derived relaxing factor from isolated human arteries. J. Hypertens., 3 (Suppl.), S97–S99.
Thomson, A.J., Lunan, B.C., Cameron, A.D. et al. (1997) Nitric oxide donors induce ripening of the human uterine cervix: a randomised controlled trial. Br. J. Obstet. Gynaecol., 104, 1054–1057.[Web of Science][Medline]
Van Voorhis, B.J., Dunn, M.S., Snyder, G.D. et al. (1994) Nitric oxide: an autocrine regulator of human granulosa-luteal cell steroidogenesis. Endocrinology, 135, 1799–1806.[Abstract]
Van Voorhis, B.J., Moore, K., Strijbos, P.J.L.M. et al. (1995) Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo. J. Clin. Invest., 96, 2719–2726.
Vega, M., Johnson, M.C., Díaz, H.A. et al. (1998) Regulation of human luteal steroidogenesis in vitro by nitric oxide. Endocrine, 8, 185–191.[Web of Science][Medline]
Wiltbank, M.C., Belfiore, C.J. and Niswender, G.D. (1993) Steroidogenic enzyme activity after acute activation of protein kinase (PK) A and PKC in ovine small and large luteal cells. Mol. Cell. Endocrinol., 97, 1–7.[Web of Science][Medline]
Wiltbank, M.C., Gallagher, K.P., Christensen, A.K. et al. (1990) Physiological and immunocytochemical evidence for a new concept of blood flow regulation in the corpus luteum. Biol. Reprod., 42, 139–149.[Abstract]
Zackrisson, U., Mikuni, M., Wallin, A. et al. (1996) Cell-specific localization of nitric oxid synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation. Hum. Reprod., 11, 2667–2673.
Submitted on June 22, 1999; accepted on January 27, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K. Shirasuna, S. Watanabe, T. Asahi, M. P B Wijayagunawardane, K. Sasahara, C. Jiang, M. Matsui, M. Sasaki, T. Shimizu, J. S Davis, et al. Prostaglandin F2{alpha} increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis Reproduction, April 1, 2008; 135(4): 527 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. V. Orozco, Z. Sosa, V. Fillipa, F. Mohamed, and A. M. Rastrilla The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide in oestrus day rats: characterization of an ex vivo system J. Endocrinol., December 1, 2006; 191(3): 587 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sen, E. Choudhary, E. K. Inskeep, and J. A. Flores Effects of Selective Protein Kinase C Isozymes in Prostaglandin2{alpha}-Induced Ca2+ Signaling and Luteinizing Hormone-Induced Progesterone Accumulation in the Mid-Phase Bovine Corpus Luteum Biol Reprod, April 1, 2005; 72(4): 976 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ohgami, I. Ilieva, K. Shiratori, Y. Koyama, X.-H. Jin, K. Yoshida, S. Kase, N. Kitaichi, Y. Suzuki, T. Tanaka, et al. Anti-inflammatory Effects of Aronia Extract on Rat Endotoxin-Induced Uveitis Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 275 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Klipper, T. Gilboa, N. Levy, T. Kisliouk, K. Spanel-Borowski, and R. Meidan Characterization of endothelin-1 and nitric oxide generating systems in corpus luteum-derived endothelial cells Reproduction, October 1, 2004; 128(4): 463 - 473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Shiratori, K. Ohgami, I. B. Ilieva, Y. Koyama, K. Yoshida, and S. Ohno Inhibition of Endotoxin-Induced Uveitis and Potentiation of Cyclooxygenase-2 Protein Expression by {alpha}-Melanocyte-Stimulating Hormone Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 159 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ohgami, K. Shiratori, S. Kotake, T. Nishida, N. Mizuki, K. Yazawa, and S. Ohno Effects of Astaxanthin on Lipopolysaccharide-Induced Inflammation In Vitro and In Vivo Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2694 - 2701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Jaroszewski, D. J. Skarzynski, R. M. Blair, and W. Hansel Influence of Nitric Oxide on the Secretory Function of the Bovine Corpus Luteum: Dependence on Cell Composition and Cell-to-Cell Communication Exp Biol Med, June 1, 2003; 228(6): 741 - 748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Tamanini, G. Basini, F. Grasselli, and M. Tirelli Nitric oxide and the ovary J Anim Sci, February 1, 2003; 81(14_suppl_2): E1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Komar and T. E. Curry Jr Localization and Expression of Messenger RNAs for the Peroxisome Proliferator-Activated Receptors in Ovarian Tissue from Naturally Cycling and Pseudopregnant Rats Biol Reprod, May 1, 2002; 66(5): 1531 - 1539. [Abstract] [Full Text]
|
|
|
|
 |
|
 S.-J. Tsai, M.-H. Wu, P.-C. Chuang, and H.-M. Chen Distinct regulation of gene expression by prostaglandin F2{{alpha}} (PGF2{{alpha}}) is associated with PGF2{{alpha}} resistance or susceptibility in human granulosa-luteal cells Mol. Hum. Reprod., May 1, 2001; 7(5): 415 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.B. Motta, A. Estevez, T. Tognetti, M.A.F. Gimeno, and A.M. Franchi Dual effects of nitric oxide in functional and regressing rat corpus luteum Mol. Hum. Reprod., January 1, 2001; 7(1): 43 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vega, L. Urrutia, G. Iniguez, F. Gabler, L. Devoto, and M.C. Johnson Nitric oxide induces apoptosis in the human corpus luteum in vitro Mol. Hum. Reprod., August 1, 2000; 6(8): 681 - 687. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||