TY - JOUR
AU - Rossmanith, Winfried G.
AB - Abstract Gonadotrophin releasing hormone (GnRH) plays an important regulatory role in the function and growth of human placenta, but its placental expression sites and co-localization with GnRH receptor (GnRH-R) are not well known. GnRH and GnRH-R expression has been found in both placenta and cultured trophoblasts; however, cultured trophoblastic cells very quickly lose GnRH-R message and subsequently receptor protein. Speculating that endogenously released GnRH induced this down-regulation, we determined GnRH-R in cultures of trophoblastic cells in the presence of a neutralizing anti-GnRH antibody. Cells incubated with this antibody showed a strong signal for the GnRH-R, while those cultured without did not (as analysed by immunofluorescence, reverse transcription-polymerase chain reaction, protein `dot blot' and Western blotting). Furthermore, addition of the GnRH agonist buserelin led to a reduction of the receptor protein. We have therefore shown that GnRH released from trophoblastic cells down-regulates GnRH-R in these trophoblastic cells. Having previously shown that trophoblast layers were synchronously positive for GnRH and GnRH-R, these new findings support the hypothesis of an ultrashort feedback regulation of trophoblasts by GnRH involving autocrine regulation of GnRH-R. cell culture, down-regulation, GnRH, GnRH receptor, placenta Introduction During pregnancy, the hypothalamic-pituitary-gonadal axis is markedly influenced by the actions of placental hormones. The suppression of pituitary responses to gonadotrophin-releasing hormone (GnRH) and reduction in circulating levels of immunoreactive GnRH and LH are attributable to the feedback effects of placental steroid and neuroendocrine hormones (Rubinstein et al., 1978; Petraglia et al., 1996). Recently, we reported that human first trimester and term placentae express GnRH and GnRH receptor (GnRH-R) (Wolfahrt et al., 1998). Exogenous GnRH had earlier been found to stimulate human chorionic gonadotrophin (HCG) production and to be released from placental tissue (Siler Khodr and Khodr, 1981; Szilagyi et al., 1992). These findings suggest that the placenta may possess GnRH-mediated regulatory systems, presumably autocrine mechanisms. Evidence for ultrashort feedback regulation of GnRH in normal hypothalamic tissues has preceded molecular characterization of the human GnRH-R (Hyyppa et al., 1971; Valenca et al., 1987) and has been obtained using immortalized neuronal cells (Krsmanovic et al., 1993; for review see Kalra and Kalra, 1994). Very recent evidence has also pointed to direct effects of GnRH as an activator of the GnRH-R promoter (Lin and Conn, 1999; Maya-Nunez and Conn, 1999). While stimulation of HCG gene expression by GnRH has not been analysed at the molecular level, the LH-beta gene has been found to be activated by GnRH (Dorn et al., 1999; Tremblay and Drouin, 1999). In order to show that trophoblastic cells, which have been shown to express GnRH-R, are sensitive to the actions of GnRH, we initiated experiments with cultured trophoblasts and have analysed their response to GnRH. The present paper deals with the observation of GnRH-R down-regulation during culture and inhibition of this down-regulation by capturing endogenously secreted GnRH with the help of a neutralizing anti-GnRH antibody. Materials and methods Characterization of tissues used The experiments were conducted with human placentae of different gestational ages. Permission to use human material was granted by the local Ethical Committee and informed consent was obtained from the patients prior to the study. Fresh human placentae were obtained immediately following spontaneous vaginal deliveries, Caesarean sections, or following therapeutic abortion at first trimester of gestation. The placental tissue was placed on ice; fragments (5–30 g) were dissected from the placentae and adherent membranes were cut off. The tissue blocks were then washed in physiological saline (0.9%) and subjected to further investigation. All assays were performed with three or more different placentae. Cultures of trophoblastic cells Suspensions of human trophoblast cells were prepared using a protocol as previously described (Li et al., 1996). Following the mechanical and enzymatic dispersal of placental fragments, cells were washed in Ham's F-12/Dulbecco's modified Eagle's medium (DMEM; Sigma, Munich, Germany) and then separated according to their sizes by sedimentation into bovine serum albumin density gradient (1, 2 and 3% BSA, 1 h by gravity). The trophoblasts at the bottom of the tube were incubated with a mixture of monoclonal mouse antibodies against HLA-ABC (w6/32) and HLA-DR/DP/DQ (CR3-43; both from Dako). Afterwards they were washed and treated with sheep anti-mouse antibodies coupled to magnetic beads (Dynal, Hamburg, Germany). Cells positive for HLA antigens were retained in a magnetic field while HLA-negative cells could be pipetted away. These latter cells were then used for further experiments. Purified cells were seeded onto collagen-coated slides or culture flasks (Becton Dickinson, Heidelberg, Germany). Cells were cultured in Ham's F-12/DMEM containing 17% fetal bovine serum (Boehringer, Mannheim, Germany) at 37°C in a humidified atmosphere with 5% CO2 for 1-7 days. Alternatively, cells were cultured serum-free according to a previously published method (Li et al., 1996). In-situ reverse transcription-polymerase chain reaction (RT–PCR) In-situ RT-PCR in human first and third trimester trophoblastic cells was performed as previously described (Wolfahrt et al., 1998). Single trophoblasts, from monolayers cultured for up to 3 days, were treated with proteinase K. Following reverse transcription with GnRH- or GnRH-R-specific primers (Wolfahrt et al., 1998), PCR was performed with intron-spanning primers to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP primers and nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) was used for substrate visualization (Wolfahrt et al., 1998). RT-PCR RNA isolation, reverse transcription, primers and nested PCR conditions have been previously described (Wolfahrt et al., 1998). MCF-7, a breast adenocarcinoma cell line (ATCC HTB 22; American Type Culture Collection, Rockville, MD, USA), was used as positive control. Cell culture modulations To demonstrate possible GnRH receptor downregulation, trophoblast cells adherent to the slides were incubated with medium alone (see above), with anti-GnRH antibody, or anti-GnRH antibody plus 10–4 or 10–8 mol/l buserelin (Hoechst, Frankfurt/Main, Germany). We used the anti-GnRH antibody (DSIL-LHRH-A1) produced by a hybridoma cell line (Silversides et al., 1985; ATCC). The cell culture supernatant containing GnRH antibodies was concentrated by centrifugation through Centricon Plus-80 centrifugal filter devices (Millipore, Eschborn, Germany). Different preparations of this cell culture supernatants were adjusted to equal anti-GnRH antibody concentrations using a sandwich enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. Rabbit anti-mouse Ig (Dako, Hamburg, Germany) in carbonate buffer (50 mmol/l pH 9.6) was coated onto microtitre plates (Dynex, Stuttgart, Germany); plates were blocked with 1% BSA in phosphate-buffered saline (PBS). The concentrated culture supernatants were titrated into these plates. Biotinylated GnRH [prepared according to the description of Prakash et al. (1998)] was then added and its binding was detected by streptavidin-peroxidase (Amersham Pharmacia Biotech, Freiburg, Germany) and subsequent ortho-phenylen-diamine conversion. For control purposes, fresh culture medium was concentrated similarly and added to the trophoblast cultures. The medium was changed daily. Cells were harvested after 7 days in culture, used to prepare membrane fractions, or processed for immunofluorescence after fixation for 10 min in 4% paraformaldehyde PBS solution. Preparation of anti-GnRH-R antibodies To obtain anti-GnRH-R antibodies, rabbits were immunized with a synthetic peptide corresponding to amino acids 5–24 (ASPEQNQNHCSAINNSIPLM) of human GnRH-R conjugated to thyroglobulin (Bauminger and Wilchek, 1980). Immunization was performed according to standard protocols, i.e. after collecting preimmune sera, the antigen was applied, and 28 days after the initial immunization booster injections were given at weekly intervals. Antibody titres were tested by an ELISA using the peptide as antigen. The peptide (dissolved in dimethylsulphoxide) was coupled to microtitre plates. Plates were blocked by 1% BSA in PBS. Titrations of the antisera were then added. Binding was detected using goat anti-rabbit Ig peroxidase conjugate (Dako) and ortho-phenylene-diamine as substrate. Immunofluorescence (IFL) All steps of the immunofluorescence staining were performed at room temperature in a humid and dark chamber. After equilibration in PBS, slides were blocked with goat serum for 10 min. They were then incubated with rabbit anti-GnRH receptor antiserum or with preimmune serum diluted 1/2000 in PBS. After washing in PBS, slides were incubated with biotinylated goat anti-rabbit (Ig) antibodies. Binding was developed using fluorescein-coupled avidin (Vector Laboratories, Wiesbaden, Germany). Alternatively, staining for GnRH was performed using the mouse anti-GnRH antibody (DSIL-LHRH-A1; see above) and goat anti-mouse Ig fluorescein (Dianova, Hamburg, Germany). The slides were finally coverslipped with mounting medium (Vectashield Mounting Medium, Vector Labaratories). Fluorescence was visualized by means of a stereo fluorescence microscope (Zeiss, Oberkochen, Germany). Dot blots Membrane fractions of the human trophoblasts were prepared as previously described (Karande et al., 1995). Briefly, the cells were homogenized on ice in buffer [20 mmol/l Tris–HCl pH 8.0, 150 mmol/l NaCl, 1 mmol/l CaCl2, 1 mmol/l protease inhibitor cocktail (Sigma)]. After removing cell debris by centrifugation at 800 g, the homogenate was centrifuged for 4 h at 100 000 g pelleting the membrane fraction. The pellet was solubilized in 5 mmol/l 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) in 10 mmol/l Tris–HCl buffer, pH 7.4. Protein concentrations were assayed by the BCA Protein Assay Kit (Pierce, Rockford, IL, USA), using BSA as standard. Quantities (500 ng) of membrane extract were blotted onto nitrocellulose filters (Hybond-C Super; Amersham Pharmacia Biotech) which were then blocked in 3% BSA/PBS containing 1% goat serum. After incubation with either GnRH-R antiserum or preimmune serum in a dilution of 1:3000, the membranes were exposed to goat anti-rabbit Ig coupled to peroxidase (Dako, Hamburg, Germany). Immunoreactive dots were visualized by chemiluminescence (ECL Kit; Amersham Pharmacia Biotech). Western blotting The same membrane fractions as used above were separated by sodium dodecyl sulphate-polyacrylamide (SDS–PAGE) in a 10% polyacrylamide gel under reducing conditions. A prolactin-releasing pituitary rat cell line (GH3; ATTC) served as negative control. 30 μg membrane extract was separated by PAGE and electrotransferred on to nitrocellulose (Towbin et al., 1979). Coloured marker proteins (Rainbow markers; Amersham Pharmacia Biotech) were used to analyse molecular weights. In addition to the anti-GnRH-R antiserum described above, we used a commercial anti-GnRH-R monoclonal antibody [clone F1G4; Dunn, Asbach, Germany (Karande et al., 1995)]. After incubation of membranes with these antibodies, their binding was detected by using peroxidase conjugated to rabbit anti-mouse IgG. Immunoreactive bands were visualized by chemiluminescence (ECL Kit). Results Analysis of GnRH-R mRNA in cultured trophoblastic cells We have previously reported that by in-situ RT-PCR, mRNA for GnRH and its receptor could be found in the trophoblast layers of human placentae (Wolfahrt et al., 1998). However, whereas in freshly isolated trophoblasts we were able to detect mRNA for GnRH, we have repeatedly failed to detect the mRNA for the GnRH receptor (Figure 1). Negative controls for these in-situ RT-PCR (for the absence of genomic DNA amplification and further contaminations) have been published earlier (Wolfahrt et al., 1998). The staining of placental tissues in that publication serves as positive control. By nested RT-PCR with isolated RNA, we detected GnRH-R message from freshly prepared trophoblastic cells (Figure 2; lane 3). However, in cells cultured for 24 h, the signal was barely visible (lane 4). Glyceraldehyde-3-phosphate dehydrogenase signals [tested as a housekeeping gene (Dahia et al., 1997)] were of equal intensities in these samples (data not shown). Since the GnRH-R is most probably necessary to mediate GnRH effects on HCG synthesis and secretion, we started to analyse the presence and regulation of the GnRH-R polypeptide in the human placenta. GnRH and GnRH-R immunofluorescence in trophoblastic cells A rabbit serum raised against an N-terminal 21 amino acid peptide (P5-24) of the GnRH receptor recognized the trophoblast layers of placentae and freshly isolated placental trophoblastic cells. The preimmune serum at the same dilution did not cause staining. Whereas cells in culture for 24 h exhibited strong GnRH-R staining in all cells (Figure 3A), cells which were cultured for prolonged periods always showed a gradual loss of GnRH-R staining (Figure 3B). In contrast to these observations, GnRH staining remained high after prolonged culture periods (Figure 3C,D). Since it has been shown that GnRH triggers down-regulation of the GnRH-R in a pituitary cell type (Mason et al., 1994), we reasoned that endogenously secreted GnRH may be causing this receptor protein loss. After detecting GnRH mRNA in these cultured trophoblasts (Figure 1A), the peptide was shown by immunofluorescense in trophoblasts (Figure 3C,D), and found to be released into the culture supernatant, by an ELISA which measured secreted GnRH (Kleine et al., 2000). With the presence of GnRH in the culture supernatants having been confirmed, we added a neutralizing anti-GnRH monoclonal antibody to the cultures. Trophoblasts in culture for 7 days without the anti-GnRH antibody showed the earlier observed reduction of the receptor protein (Figure 4A). In the presence of saturating doses of this neutralizing anti-GnRH antibody, however, the cells remained strongly positive for GnRH-R (Figure 4B). These findings demonstrated that GnRH was responsible for the observed receptor loss. We then added to these cultures the GnRH agonist buserelin which we have shown not to cross-react with the monoclonal anti-GnRH antibody and which should still bind to the receptor. At a 10–4 mol/l concentration, the addition of buserelin led to a reduction of the receptor protein (Figure 4C), while at a concentration of 10–8 mol/l it had no effect (not shown). These results provided evidence that the antibody in the culture, besides capturing the soluble GnRH, did not affect the receptor which still could be down-regulated by the GnRH agonist. Inhibition of GnRH-R mRNA loss by the anti-GnRH antibody We also demonstrated by RT-PCR that in trophoblastic cells cultured for 24 h, the presence of the anti-G GnRH antibody prevented the loss of signal for the GnRH-R message (Figure 2; lane 5), suggesting regulation of GnRH-R at the transcriptional level. Dot blot analysis of GnRH-R down-regulation The results of the immunofluorescence studies were confirmed by dot blot analysis using the membrane fraction of cultured trophoblasts (Figure 5). Absence of the primary antibody (incubation with preimmune serum as negative control for the dot blot assay) showed no signal, whereas by use of the anti-GnRH-R antiserum (right blot) the following results were obtained: trophoblasts in culture for 1 day showed a signal for the GnRH-R polypeptide, while with trophoblasts after 7 days in culture the dot was barely visible. After culturing the cells in the presence of GnRH antibody for 7 days, a strong signal for the GnRH-R polypeptide was observed. Addition of 10–4 mol/l buserelin to the culture led to a reduction of the receptor protein staining, while a concentration of 10–8 mol/l buserelin actually resulted in an enhanced signal for GnRH-R. Since we wanted to exclude the possibility that components of the concentrated culture medium were responsible for our findings, fresh culture medium was concentrated to a similar extent as the anti-GnRH antibody containing culture supernatants. This concentrated medium did not inhibit receptor down-regulation, proving that the antibody secreted from the hybridoma cell line was responsible for the inhibition of GnRH-R down-regulation. Western blot analysis of GnRH-R expression In order to confirm that the signals observed were derived from the receptor protein, we performed Western blotting with membrane preparations of cultured trophoblastic cells. The results obtained were not expected to be quantitative due to the saturation levels of protein used in each lane. For the anti-GnRH-R antibody, we employed the F1G4 monoclonal antibody, which has been reported to stain GnRH-R in Western blots (Karande et al., 1995). Two intensely stained bands of the expected size (~66 and 56 kDa) were present in all solubilized human trophoblast membrane preparations (Figure 6). As a negative control, we employed prolactin-releasing pituitary cells (GH3; lane 1), since prolactinoma tumours were the only cell type consistently negative for GnRH-R (Sanno et al., 1997). These cells did not reveal any band. In human trophoblasts cultured for 1 or 7 days without additions to the culture medium, the 66 and 56 kDa bands were visible (lane 2 and 3), whereas trophoblasts cultured in presence of anti-GnRH antibodies plus/minus buserelin displayed an additionally 30 kDa band (lane 4–6). We excluded the possibility that the 30 kDa signal was derived from the light chain of the anti-GnRH antibody which might have contaminated the cell membrane preparations as the 30 kDa band was not visible in the absence of any anti-GnRH-R antibody. This showed that the 30 kDa band could only be detected by the anti-GnRH-R antibody F1G4 and not by the anti mouse Ig reagent alone. Discussion The present study deals with the functional interaction of GnRH and its cognate receptor in isolated human trophoblastic cells. It is part of a larger study investigating hormonal regulation in the human placenta functions and the involvement of ultrashort feedback mechanisms. We have demonstrated by four independent methods (immunofluorescence, RT-PCR, `dot blot' and Western blotting) that GnRH in the culture medium leads to reduced receptor levels on the cultured trophoblastic cells. This was demonstrated using an anti-GnRH antibody which we have shown to bind to natural GnRH, but not to analogues such as [d-Lys6]GnRH, buserelin, or [des-Gly10]GnRH (Kleine et al., 2000). The antibody could, however, block functional interactions of GnRH with its receptor. The result of this lack of interaction on the cultured cells was that GnRH-R was maintained on the cells, while in the absence of this neutralizing antibody, the receptor polypeptide was mostly lost. A control medium never exposed to the hybridoma cells, and concentrated in the same way, showed that antibodies in the hybridoma supernatant and not medium components were responsible for the inhibition of receptor down-regulation. By analysing the time course of this reduced receptor expression, we found that GnRH-R mRNA was barely detected by sensitive nested RT-PCR after 24 h, whereas the receptor polypeptide was easily detected for up to 72 h but was down-regulated after 4-7 days. We have not yet attempted to render the nested RT-PCR analysis completely quantitative. However, since the samples were treated with identical reagents and similar aliquots used under identical conditions, we took these results as a hint that the anti-GnRH antibodies might influence GnRH-R expression at the RNA level. One of the reasons not to proceed at the quantitative level was continuous problems with placental GnRH-R cDNA amplification, as previously reported (Santra et al., 2000). There are reports that GnRH reduces GnRH-R concentrations in ovine pituitary cultures as well as in a pituitary cell line, αT3-1 (Wu et al., 1994; Kakar et al., 1997). On the other hand, it has been reported that GnRH induces GnRH-R transcription (Norwitz et al., 1999), again in the αT3-1 cells. An interesting variable in the down- or up-regulation process might be the availability of GnRH: while constant treatment of cells with GnRH leads to receptor loss, pulsatile application may enhance its levels. The paper cited by Maya-Nunez and Conn (1999), however, does not support this speculation. The high pulsatile GnRH doses applied by Adams et al. (1996) also led to receptor down-regulation, possibly because of oestradiol withdrawal. This point remains to be clarified. Interestingly, Western blot analysis showed a 30 kDa immunoreactive band when the cultures were treated with anti-GnRH antibody. Since this molecule was only found under conditions where the receptor was present due to GnRH withdrawal, whereas no immunoreactivity was present when the receptor was down-regulated by GnRH, we do not think that the 30 kDa band represents a degradated receptor. It may represent an alternatively spliced receptor similar to one previously reported (Grosse et al., 1997). A band of similar size has also been found (Santra et al., 2000). Sequencing of the product is underway in our laboratory. Recently, Leung and colleagues (Cheng et al., 2000) published the sequence of a full length placental GnRH-R mRNA identical to the one previously obtained from the pituitary (Kakar et al., 1992; Chi et al., 1993). These authors also used semiquantitative RT–PCR to study GnRH-R expression in cultured extravillous trophoblast (EVT). Their results in the mRNA analysis are in contrast to those data reported here, since they have found stimulation of a GnRH-R by GnRH agonist in their experiments. The obvious differences between these findings may be explained by the use of different cell preparations. Cheng et al. used first trimester extravillous primary cultures, whereas in most of the assays we used third trimester villous trophoblastic cell preparations depleted of EVT cells using anti-HLA magnet beads, reasoning that EVT express HLA-C and HLA-G (King et al., 1996). Furthermore, the [d–Ala6]-GnRH agonist used by Cheng et al. (2000) might react with the GnRH-R differently from endogenously produced GnRH or buserelin. We have observed by in-situ RT-PCR (Wolfahrt et al., 1998) as well as by RT-PCR with extracted RNA, that GnRH-R mRNA was rapidly and completely lost upon culturing the isolated trophoblasts, while the receptor polypeptide could still be detected 3 days later. In contrast to these results, following GnRH analogue treatment in αT3-1 cells, the GnRH-R mRNA was less reduced than the receptor polypeptide (Mason et al., 1994). One reason for this discrepancy might be the exceptional high number of GnRH-R on this cell line (Kakar et al., 1997). Our results also raise the question as to what mechanism might maintain the GnRH-R receptor expression in placental tissue since we have demonstrated its presence in almost any cell of the trophoblast layers (Wolfahrt et al., 1998). High progesterone levels in the pituitary have been shown to decrease GnRH-R mRNA, while oestradiol and inhibin have favoured its expression (Wu et al., 1994). In placenta, the predominant sex steroid is progesterone whereas oestradiol and inhibin are found to a lesser extent (Batra et al., 1979; Petraglia et al., 1996). The presence of GnRH, which we have shown to be expressed in the trophoblast, would not favour GnRH-R expression. Are there other major mechanisms that may still help in maintaining GnRH-R expression? Since the placenta expresses a plethora of neuroendocrine hormones (Petraglia et al., 1996), under conditions where no cell specialization has been found, it is possible that the activation of the GnRH-R gene might be influenced by unknown transcription factors. Activator elements in the GnRH-R gene promoter have been identified (Norwitz et al., 1999; Tremblay and Drouin, 1999; Maya-Nunez and Conn, 1999). Whether any of these might be responsible for its sustained placental expression despite the presence of down-regulatory elements is open to experimental testing. In this study, we tried to detect an ultrashort feedback mechanism regulating the neuroendocrine network in placenta. The results presented here show that such a mechanism may exist. Indeed, together with the demonstration that GnRH-R and GnRH are synthesized within the same cell, we would argue that such mechanisms are important. In summary, we provide evidence in favour of the down-regulation of GnRH-R in the placenta by its cognate ligand, GnRH. Figure 1. View largeDownload slide Localization of GnRH and GnRH receptor (GnRH-R) mRNA in human trophoblastic cells by in-situ reverse transcription-polymerase chain reaction (RT-PCR). In-situ RT-PCR was performed on trophoblasts after 24 h in culture. (A) In-situ RT-PCR with GnRH primers; (B) in-situ RT-PCR with GnRH-R primers. Positive and negative controls have been published earlier (Wolfahrt et al., 1998). The final blue black substrate deposit was detected by visual examination. White bar = 20 μm. Figure 1. View largeDownload slide Localization of GnRH and GnRH receptor (GnRH-R) mRNA in human trophoblastic cells by in-situ reverse transcription-polymerase chain reaction (RT-PCR). In-situ RT-PCR was performed on trophoblasts after 24 h in culture. (A) In-situ RT-PCR with GnRH primers; (B) in-situ RT-PCR with GnRH-R primers. Positive and negative controls have been published earlier (Wolfahrt et al., 1998). The final blue black substrate deposit was detected by visual examination. White bar = 20 μm. Figure 2. View largeDownload slide Loss of GnRH receptor (GnRH-R) mRNA in cultured trophoblastic cells. Total RNA was reversely transcribed using random hexamer primers. The cDNA was amplified using GnRH-R-specific primers (35 cycles) and reamplified with nested primers (40 cycles). Samples: M, Msp I digest of pBR322; lane 1, negative control (no cDNA template); lane 2, positive control (MCF-7); lane 3, RNA from first trimester trophoblastic cells after preparation (day 0); lane 4, first trimester trophoblastic cells cultured for 24 h; lane 5, first trimester trophoblastic cells cultured for 24 h in the presence of monoclonal anti-GnRH antibody. Figure 2. View largeDownload slide Loss of GnRH receptor (GnRH-R) mRNA in cultured trophoblastic cells. Total RNA was reversely transcribed using random hexamer primers. The cDNA was amplified using GnRH-R-specific primers (35 cycles) and reamplified with nested primers (40 cycles). Samples: M, Msp I digest of pBR322; lane 1, negative control (no cDNA template); lane 2, positive control (MCF-7); lane 3, RNA from first trimester trophoblastic cells after preparation (day 0); lane 4, first trimester trophoblastic cells cultured for 24 h; lane 5, first trimester trophoblastic cells cultured for 24 h in the presence of monoclonal anti-GnRH antibody. Figure 3. View largeDownload slide Detection of GnRH and GnRH receptor (GnRH-R) by immunofluorescence. Single cell preparations from human placenta were cultured for 24 h or 7 days on collagen-coated slides and incubated with anti-GnRH-R or anti-GnRH antibodies. Anti-GnRH-R binding was revealed using biotinylated goat anti-rabbit (Ig) antibodies and avidin-fluorescein, and anti-GnRH binding by goat anti-mouse Ig fluorescein. (A, B) GnRH-R staining in trophoblasts cultured for 1 day (A) or 7 days (B); (C, D) GnRH staining in cells cultured for 1 day (C) or 7 days (D) Figure 3. View largeDownload slide Detection of GnRH and GnRH receptor (GnRH-R) by immunofluorescence. Single cell preparations from human placenta were cultured for 24 h or 7 days on collagen-coated slides and incubated with anti-GnRH-R or anti-GnRH antibodies. Anti-GnRH-R binding was revealed using biotinylated goat anti-rabbit (Ig) antibodies and avidin-fluorescein, and anti-GnRH binding by goat anti-mouse Ig fluorescein. (A, B) GnRH-R staining in trophoblasts cultured for 1 day (A) or 7 days (B); (C, D) GnRH staining in cells cultured for 1 day (C) or 7 days (D) Figure 4. View largeDownload slide Inhibition of GnRH receptor (GnRH-R) down-regulation by anti-GnRH antibodies. Cells were cultured for 7 days in the presence of medium alone (A), anti-GnRH antibody (B), or anti-GnRH antibody plus 10–4 mol/l buserelin (C). Subsequent staining with rabbit anti-GnRH-R antibody plus biotinylated goat anti-rabbit Ig plus avidin-fluorescein showed the inhibition of GnRH receptor down-regulation by an antibody against endogenously secreted GnRH. Figure 4. View largeDownload slide Inhibition of GnRH receptor (GnRH-R) down-regulation by anti-GnRH antibodies. Cells were cultured for 7 days in the presence of medium alone (A), anti-GnRH antibody (B), or anti-GnRH antibody plus 10–4 mol/l buserelin (C). Subsequent staining with rabbit anti-GnRH-R antibody plus biotinylated goat anti-rabbit Ig plus avidin-fluorescein showed the inhibition of GnRH receptor down-regulation by an antibody against endogenously secreted GnRH. Figure 5. View largeDownload slide Dot blot analysis of GnRH receptor (GnRH-R) expression. To demonstrate GnRH-R down-regulation, trophoblast cells were cultured with medium alone, with GnRH antibody, or with GnRH antibody plus 10–4 or 10–8 mol/l buserelin (as indicated). Membrane fractions were blotted onto nitrocellulose (500 ng each sample: the GnRH-R peptide 5–24 was included as positive control). GnRH-R immunoreactive dots were visualized by chemiluminescence. Left blot: incubation with preimmune rabbit serum as negative control; right blot: incubation with rabbit anti-GnRH-R serum as primary antibody. The medium control contained no anti-GnRH antibody. Figure 5. View largeDownload slide Dot blot analysis of GnRH receptor (GnRH-R) expression. To demonstrate GnRH-R down-regulation, trophoblast cells were cultured with medium alone, with GnRH antibody, or with GnRH antibody plus 10–4 or 10–8 mol/l buserelin (as indicated). Membrane fractions were blotted onto nitrocellulose (500 ng each sample: the GnRH-R peptide 5–24 was included as positive control). GnRH-R immunoreactive dots were visualized by chemiluminescence. Left blot: incubation with preimmune rabbit serum as negative control; right blot: incubation with rabbit anti-GnRH-R serum as primary antibody. The medium control contained no anti-GnRH antibody. Figure 6. View largeDownload slide Western blot analysis of GnRH receptor (GnRH-R) expression. 30 μg of membrane preparations were separated by reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis and blotted onto Hybond C membranes. These were incubated with a mouse anti-GnRH-R monoclonal antibody. Binding was detected by a rabbit anti-mouse Ig peroxidase conjugate and chemiluminescence. The results obtained were not expected to be quantitative due to saturation levels of protein in each lane. Samples: prolactin-secreting GH3 cells (lane 1, negative control); trophoblasts cultured for 24 h (lane 2); trophoblasts cultured for 7 days (lane 3); trophoblasts cultured for 7 days with anti-GnRH antibody (lane 4); same as in lane 4 plus 10–4 mol/l buserelin (lane 5); same as in lane 4 plus 10–8 mol/l buserelin (lane 6). Arrows indicate size marker proteins. Figure 6. View largeDownload slide Western blot analysis of GnRH receptor (GnRH-R) expression. 30 μg of membrane preparations were separated by reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis and blotted onto Hybond C membranes. These were incubated with a mouse anti-GnRH-R monoclonal antibody. Binding was detected by a rabbit anti-mouse Ig peroxidase conjugate and chemiluminescence. The results obtained were not expected to be quantitative due to saturation levels of protein in each lane. Samples: prolactin-secreting GH3 cells (lane 1, negative control); trophoblasts cultured for 24 h (lane 2); trophoblasts cultured for 7 days (lane 3); trophoblasts cultured for 7 days with anti-GnRH antibody (lane 4); same as in lane 4 plus 10–4 mol/l buserelin (lane 5); same as in lane 4 plus 10–8 mol/l buserelin (lane 6). Arrows indicate size marker proteins. 3 To whom correspondence should be addressed at: Department of Gynaecology and Obstetrics, Diakonissenkrankenhaus Karlsruhe Diakonissenstr. 28, D-76199 Karlsruhe, Germany. E-mail: rossmanith@diak-ka.de We wish to thank Mrs Maria Metten, Department of Experimental Endocrinology, University of Göttingen, for expert technical assistance. We express our gratitude to Professor Ashley Grossman, St Bartholomew's Hospital, London, UK, for his editorial comments and suggestions. This work has been supported by a grant to W.G.R. from the Deutsche Forschungsgemeinschaft (DFG Ro657/6-4). References Adams, B.M., Sakurai, H. and Adams, T.E. 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TI - Endogenous regulation of the GnRH receptor by GnRH in the human placenta
JF - Molecular Human Reproduction
DO - 10.1093/molehr/7.1.89
DA - 2001-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/endogenous-regulation-of-the-gnrh-receptor-by-gnrh-in-the-human-ddfhAdK0x9
SP - 89
EP - 95
VL - 7
IS - 1
DP - DeepDyve
ER -