Molecular Human Reproduction, Vol. 6, No. 3, 264-268,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
The progesterone receptor and ubiquitin are differentially regulated within the endometrial glands of the natural and stimulated cycle
1 Department of Obstetrics & Gynaecology, University of Sydney, Westmead Hospital, Sydney, NSW 2145, Australia, and 2 School of Human Development and 3 School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, UK
Abstract
The initiation of human pregnancy requires precisely timed development of the endometrium to receive the implanting blastocyst. The ovarian steroid hormones are essential for development and maintenance of a hospitable uterine environment. The hormonal regimes employed in assisted reproduction procedures are known to alter the abundance of specific endometrial receptors for these steroids. Since, in the presence of ligand, the progesterone receptor (PR) is known to be modified by the small intracellular protein ubiquitin, we have investigated the localization of ubiquitin and PR within the endometrial glands of 28 fertile women during a monitored menstrual cycle and also during a stimulated cycle prior to oocyte donation. We have also observed the number of gland cells undergoing cell division as demonstrated by the presence of Ki67 immunostaining. We demonstrate that the percentage of ubiquitin-positive nuclei increases from day four post-ovulation to day 10 post-ovulation in the natural cycle, but that this increase is not seen during a stimulated cycle. The presence of PR within glandular epithelium and the proliferation of gland cells were only observed during the early secretory phase and did not appear to vary significantly between the two cycles. We conclude that ubiquitin may play an important role in endometrial development and that perturbation of ubiquitin may be related to the lower implantation rate seen in the stimulated cycle.
IVF/natural cycle/progesterone receptor/stimulated cycle/ubiquitin
Introduction
Donated human eggs have been in clinical use for the treatment of infertility since 1984, when Lutjen et al. reported the first successful pregnancy resulting from oocyte donation, IVF and embryo transfer (Lutjen et al., 1984
). In contrast to standard IVF procedures (where the female is stimulated by administration of exogenous gonadotrophins to produce a supra-physiological number of mature eggs at the time of treatment), oocyte recipients may undergo embryo transfer during a natural or programmed menstrual cycle. In the latter instance, the amounts of steroid hormones administered are still much less than the concentrations observed in stimulated cycles.
The sharing of oocytes between donors and recipients enables comparisons to be made whilst controlling for oocyte quality. In one such study, where the quality of embryos transferred and age of patients were matched, the implantation rate in the donors was found to be significantly less than that in the recipients (Check et al., 1992). This difference in implantation rate may be due to the effects of the clomiphene citrate/human menopausal gonadotrophin (HMG)/human chorionic gonadotrophin (HCG) regimen administered on endometrial development. However, another explanation for this could be that oocyte donors may have underlying endometrial defects, which would be less common in oocyte recipients.
Implantation of the human embryo may only occur within a tightly regulated `window of implantation' on days 6–10 post-ovulation. A number of studies have attempted to date the endometrium in the stimulated cycle (Garcia et al., 1984; Forman et al., 1989; Navot et al., 1989; Sharma et al., 1990; Ben-Nun et al., 1992; Seif et al., 1992; Macrow et al., 1994; Kolb and Paulson, 1997). It appears that shortly after ovulation induction the endometrial stroma is advanced in development (Garcia et al., 1984; Forman et al., 1989; Kolb and Paulson, 1997), particularly in terms of premature spiral artery formation (Seif et al., 1992; Macrow et al., 1994), while endometrial glands may be retarded in comparison to those of control groups (Navot et al., 1989; Seif et al., 1992).
The circulating ovarian hormones, oestrogen and progesterone, act through specific nuclear receptors to induce differentiation of the endometrium and to maintain an environment suitable for implantation. Progesterone receptor (PR) levels may be up-regulated by oestrogens and down-regulated by the presence of progesterone (Kreitmann et al., 1979). Increased serum steroid hormone concentrations following ovarian stimulation may, therefore, have profound effects on endometrial steroid hormone receptor density, and indeed there is evidence that ovulation induction may affect the level of steroid receptors present within endometrial epithelial or stromal cells (Hadi et al., 1994). This may be a key factor in the reduced implantation rate seen following ovulation induction.
Ubiquitin is a small regulatory protein involved in the covalent tagging of intracellular proteins to mark them for proteolysis (reviewed in Hershko and Ciechanover, 1998). Following post-translational addition of ubiquitin (ubiquitylation), proteins are degraded by a large cytosolic protease, the 26S proteasome (Hough et al., 1987). In addition to the ubiquitylation of cytoplasmic proteins, ubiquitin may have a number of roles within the cell nucleus. Both cyclins (Glotzer et al., 1991) and histones (Goldknopf and Busch, 1975) are known substrates for ubiquitylation, although in the case of histones, not necessarily for ubiquitin-mediated degradation. Several receptors may also be modified by ubiquitin, and recent reports have demonstrated ligand-mediated ubiquitylation of both the rat uterine oestradiol receptor (Nirmala and Thampan, 1995) and the chicken oviduct PR (Syvälä et al., 1998).
We have previously demonstrated the presence of anti-ubiquitin immunoreactivity within nuclei of human glandular epithelial cells of the late secretory phase endometrium (Bebington et al., 1999). The purpose of this study was to investigate whether changes in the abundance of endometrial ubiquitin were observed in women undergoing stimulation prior to assisted reproduction. To this end we quantified ubiquitin immuno-staining patterns in endometrial biopsies taken from a group of oocyte donors during the secretory phase of both a natural and a stimulated cycle. We also compared ubiquitin immunoreactivity with localization of the PR and to cell proliferation within these tissues.
Materials and methods
Sample collection
Endometrial biopsies were obtained from a total of 28 previously fertile oocyte donors (age range 25–35 years) attending clinics at the Nottingham University Research and Treatment Unit in Reproduction (NURTURE) at the Division of Reproductive Medicine, Queen's Medical Centre, Nottingham, UK, and ethical approval was also obtained from that institution. Informed consent was obtained from each patient. Tissue was collected at four time-points, at days four and 10 post-LH-surge of a monitored menstrual cycle, and also at days four and 10 after administration of HCG during the subsequent stimulated cycle. All human tissue was obtained with the approval of the Ethics Committee of NURTURE, Queen's Medical Centre, Nottingham.
Ovarian stimulation was achieved using a standard protocol combination of pituitary down-regulation with a gonadotrophin releasing hormone (GnRH) agonist (buserelin: Suprefact®, Hoechst, Hounslow, UK; Naferelin: Synarel®, Searle, High Wycombe, UK), and follicular stimulation with gonadotrophins (Humegon®: Organon, Cambridge, UK; Pergonal®: Serono, Welwyn Garden City, UK) using 2–4 ampoules daily, followed by initiation of ovulation induction, 36 h prior to oocyte retrieval, using 10 000 IU HCG (Profasi®: Serono).
Endometrial biopsies were obtained from the fundal region of the uterus using a Pipelle (Laboratoire CCD, Paris, France). Tissues were immediately fixed in 4% (w/v) paraformaldehyde and embedded in paraffin wax, allowing the generation of 4 µm sections which were cut onto glass slides pre-treated with poly-L-lysine (Sigma, Poole, UK).
A total of 62 endometrial biopsies were studied from 28 patients. Of these, 13 were obtained at day four and 12 at day 10 post-LH surge of the natural cycle (10 women were studied at both time-points). During the stimulated cycle, 19 biopsies were taken four days after HCG administration and 18 at day 10 post-HCG (16 women were studied at both time-points). Matched biopsies from six women were obtained at each of the four stages of the study and were used to study glandular epithelial cell proliferation and immunolocalization of the PR. All 62 endometrial biopsies were probed with anti-ubiquitin.
Immunocytochemistry
Sections were de-waxed in xylene and re-hydrated through a series of ethanols. Endogenous peroxidase was inactivated by treatment of sections with 0.6% (v/v) hydrogen peroxide (H2O2, Sigma), and non-specific staining was prevented by incubation of sections in 10% (v/v) normal goat serum (Dako Ltd, High Wycombe, UK) in Tris-buffered saline (TBS, 50 mmol/l Tris–HCl, 150 mmol/l NaCl, pH 7.5) for 30 min prior to immunodetection. All incubations were performed in a humidified chamber at room temperature and were followed by repeated rinses in TBS unless stated otherwise.
Anti-ubiquitin immunoreactivity was demonstrated by incubation with rabbit polyclonal antibody to ubiquitin (Dako) for 1 h, detected using biotinylated goat anti-rabbit immunoglobulin G (IgG) (Vector Laboratories, Peterborough, UK) for 30 min followed by incubation with avidin–biotinylated horseradish peroxidase complex (Vector) for 30 min.
Gland cell proliferation was demonstrated using a rabbit polyclonal antibody to a cell proliferation marker, Ki67 (Dako). Prior to immunocytochemistry, slides were immersed in 0.01 mol/l citrate buffer (pH 6.0) in a 750 W microwave oven at high power for 15 min. Bound primary antibody was detected as described for the anti-ubiquitin antibody.
The presence of PR was indicated by probing with two murine monoclonal antibodies to the human PR, recognizing either the PR-B isoform only or both PR-A and PR-B equally (Clarke et al., 1987). Antibodies were generously donated by Dr C.Clarke, Westmead Hospital, Sydney, Australia. Immunodetection followed heat-mediated antigen retrieval procedures performed in an autoclave at 121°C for 30 min in 0.01 mol/l citrate buffer, pH 6.0. Primary antibody was employed at room temperature overnight and was detected using biotinylated rabbit anti-mouse IgG (Dako) for 30 min followed by avidin–biotinylated horseradish peroxidase complex (Vector) for 30 min.
In all immunodetection protocols, bound avidin–biotinylated horseradish peroxidase was visualized using 3,3' diaminobenzidine and H2O2 (Sigma). Following antibody detection, sections were counterstained using Harris' haematoxylin (BDH, Poole, UK) and were then dehydrated and mounted in DPX (BDH).
Statistical analysis
Immunostaining was observed using bright-field microscopy. The percentage of gland cell nuclei immunopositive when probed with each antibody was observed within 300 cells counted in three randomly-chosen fields of view on each section. The data were analysed using both the paired t-test and the Wilcoxon matched-pairs signed-ranks test. Although both methods concurred at the 95% confidence interval, only the latter is presented due to the fact that we could not assume a normal distribution of the data.
Results
A proportion of glandular epithelial cells showed nuclear staining when probed with each of the antibodies employed (Figures 1–4). This immunoreactivity varied considerably with timing of the biopsy and (in the case of anti-ubiquitin immunoreactivity) also between the natural and stimulated cycles.
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No cytoplasmic reactivity was observed when tissues were probed with the anti-PR or anti-Ki67 antibodies, but considerable cytoplasmic ubiquitin was detected using rabbit anti-ubiquitin (Figure 1
). Occasional stromal cells displayed immunoreactivity with all four antibodies employed. Omission of primary antibody or substitution of non-immune serum of the same species as the primary antibody eliminated staining (data not shown).
Anti-ubiquitin immunoreactivity was observed in both nuclei and cytoplasm of the glandular epithelium as well as in occasional stromal cells (e.g. Figure 1B). In the natural menstrual cycle, an increase in the median percentage of immunopositive gland cell nuclei was seen, rising from 5% (range 0–20%) to 34% (range 1–89%) in the late secretory phase (Figure 1A and B; Figure 4A). In the stimulated cycle, however, no increase of similar magnitude was seen. Median values in the stimulated cycle were 2% (range 0–25%) and 7% (range 0–25%) at day four and day 10 following HCG administration (Figure 1C, D; Figure 4A). The density of ubiquitin immunopositive gland cell nuclei from day 10 following ovulation in the natural cycle was significantly higher than that of the stimulated cycle (P < 0.05).
PR abundance varied considerably within the tissues studied. Little or no anti-PR immunoreactivity was seen in tissue from the later stage of the secretory phase, but the majority of gland cell nuclei demonstrated immunolocalization of PR-B at day four following ovulation (Figures 2A,C; Figure 4B) with median values of 93% (range 92–97%) and 95% (range 90–98%) in the natural and stimulated cycles respectively. Total PR immunoreactivity also appeared similar in both cycles. In the early secretory phase, immunostaining was intense while only faint reactivity was observed in the late secretory tissues.
No gland cell proliferation was seen in the late secretory (day 10 post-ovulation) tissue in either the natural or stimulated cycle (Figure 3B,D and Figure 4C). A moderate number of nuclei from the early secretory phase were immunoreactive with anti-Ki67 in both cycles (Figure 3A,C and Figure 4C). The early secretory phase of the natural cycle demonstrated a median score of 8% positive cells (range 5–12%) while in the stimulated cycle a median value of 9% of glandular epithelial cells contained immunopositive nuclei (range 1–12%).
Discussion
When sharing the same batch of oocytes, the implantation rate in women subjected to the hormonal stimulation necessary for ovulation induction is reduced in comparison to oocyte recipients (Check et al., 1992). The reduced implantation rate in the stimulated cycle may be due to advanced endometrial stromal maturity accompanying retarded glandular development (Navot et al., 1989; Seif et al., 1992) or to altered abundance of endometrial steroid receptors (Hadi et al., 1994).
Ubiquitylation and ubiquitin-mediated degradation of the PR of the chicken oviduct has recently been demonstrated (Syvälä et al., 1998). We have previously shown the presence of ubiquitin within endometrial glandular epithelium throughout the menstrual cycle and in the nuclei of decidualized stromal cells in the first trimester of pregnancy (Bebington et al., 1999). The presence of nuclear ubiquitin in the endometrial glands and stroma appears to be inversely correlated with that of published observations of PR levels (Wang et al., 1994, 1998; Bebington et al., 1999). Therefore, we postulated that immunoreactivity in the stimulated cycle may indicate increased PR presence within the late secretory phase of the stimulated cycle. No such increase was detected using our techniques. Neither did we detect a decrease in PR immunoreactivity in response to stimulation, contrary to previous studies (Hadi et al., 1994). Nevertheless, our results are consistent with those of other studies investigating PR in the natural and stimulated cycle, which found little change in receptor levels (Balasch et al., 1992; Salat-Baroux et al., 1994).
Since ubiquitylation and ubiquitin-mediated proteolysis are involved in several stages of the cell cycle, we also investigated whether ubiquitin immunoreactivity was correlated to changes in the proliferation rate of these epithelial cells. Since no difference in measurable gland cell proliferation was observed between the natural and stimulated cycle, it is unlikely that the increased presence of ubiquitin within the glandular epithelium during the late secretory phase of the natural cycle is simply a reflection of increased proliferation. It is also possible that trauma generated by the initial biopsy in the early secretory phase could alter the level of immunostaining for ubiquitin observed later in the luteal phase. However, any such alteration would have been consistent for both the natural and stimulated cycle. Furthermore, a number of patients (n = 3) were only able to undergo biopsy on day 10, and the levels of ubiquitin seen within these samples were within the normal range for their study group (data not shown).
Despite the apparent low level of nuclear ubiquitin in the stimulated cycle, PR abundance is significantly reduced by day 10 after HCG administration. The mechanism of this down-regulation is not known. Increased concentrations of serum progesterone have been reported in stimulated cycles (Muasher et al., 1984) and were also noted in this investigation (data not shown). Since progesterone down-regulates PR at the level of transcription (Savouret et al., 1991), it is possible that little PR is produced and that ubiquitin-mediated down-regulation is not required in this environment. However ubiquitylation of other receptors, such as those for platelet-derived growth factor-ß and prolactin may occur (Mori et al., 1993; Cahoreau et al., 1994). These receptors are likely to be relevant to implantation and further work is planned to determine whether their expression is disrupted during the stimulated cycle.
Since implantation is considered to be the least efficient stage during the IVF-embryo transfer procedure, any alterations in endometrial morphology or activity seen in the stimulated cycle are clearly of interest. This is the first study of ubiquitin localization within the natural and stimulated human menstrual cycle. The lack of increased anti-ubiquitin immunoreactivity in glandular epithelial nuclei in the stimulated compared with the natural cycle is a novel finding. This is a particularly convincing observation as no difference in glandular PR density or cell proliferation was seen between the natural or stimulated cycle. Further studies will be necessary to determine the reason for this apparent reduction in ubiquitin concentrations and whether it is related to the lower implantation rates seen in stimulated compared with natural cycles.
Acknowledgments
The authors would like to thank staff at NURTURE and in the Department of Pathology, City Hospital, Nottingham, in particular Dr Jane Johnson, for their help in obtaining tissues. CB is supported by Westmead Fertility Centre and by a University of Nottingham Research Studentship.
Notes
4 To whom correspondence should be addressed 
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Submitted on September 7, 1999; accepted on December 13, 1999.
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