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Biology of Reproduction 62, 655-663 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

A Putative Stimulatory Role of Progesterone Acting via Progesterone Receptors in the Steroidogenic Cells of the Human Corpus Luteum1

Ulrika Ottandera, Kenichi Hosokawaa, Kui Liub, Anders Berghc, Tor Nyb, and Jan I. Olofsson2,a

a Departments of Obstetrics and Gynecology, b Medical Biochemistry and Biophysics, and c Pathology, Umeå University, S-901 87 Umeå, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To further explore the proposed auto-regulatory role of progesterone action in the human corpus luteum (CL), the expression and functional roles of progesterone receptor (PR) isoforms A and B during the luteal phase (LP) of the menstrual cycle were investigated. A total of 27 otherwise healthy patients previously scheduled for surgery were recruited after informed consent. An LH rise was detected, and CL were grouped according to age (Days 2–5 post-LH-rise, early LP; Days 6–10, mid LP; Days 11–14, late LP). Using a semiquantitative reverse transcription-polymerase chain reaction assay, the PR-B mRNA levels, which were 100- to 1000-fold lower than PR-A/B mRNA, were 46% lower (P < 0.05, n = 24) in mid LP, compared to early and late LP. CL tissue levels of progesterone and PR-A/B protein levels were inversely correlated to increasing CL age; i.e., significantly reduced levels were observed in the late LP (r2 = 0.34, P < 0.01, n = 23). Expression of PR-A/B mRNA as well as PR-A/B protein were detected by in situ hybridization and immunohistochemistry, respectively. Both methods revealed a clear and distinct localization to cells in the steroidogenic layer of the CL. Freshly obtained mid-luteal CL cells were cultured in vitro, and media were analyzed for progesterone concentrations after treatment by incremental doses of hCG and the stable PR antagonist mifepristone, alone or in combination. Mifepristone did not per se alter progesterone synthesis, but when it was added in conjunction with hCG, a dose-related inhibitory response was seen, with a maximal 47% reduction in progesterone output at a 10 µM addition (P < 0.05, n = 3). Collectively, these data implicate a stimulatory role of progesterone receptor-mediated action in the steroidogenic cells of the human CL, which may serve as an important pathway for maintaining functional homeostasis during early pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence has indicated that progesterone exerts a key role in the mammalian ovary [1–3]. In primates, recent studies suggest that progesterone is a mandatory mediator in ovulation [4, 5] and maintenance of luteal architecture [6]. The major source of progesterone synthesis in nonpregnant women is the corpus luteum (CL), a gland that produces more than ten times its own weight of progesterone during one day in the mid luteal phase (LP) [7]. In classical sex steroid-sensitive tissues, estrogen and progesterone regulate progesterone receptors (PRs). In general, estrogen enhances and progesterone decreases expression of PRs [8, 9]. However, in steroid-producing tissue such as the human CL, this signaling mechanism may be more complex, since steroid concentrations are very high. On the basis of molecular sequence data [10], two 5' flanking promoters in the single human PR gene have been identified, thereby permitting transcription of two isomers differing in size (PR-A and PR-B), with molecular weights of 94 000 and 116 000, respectively). Besides the previously most recognized function of human PR-B as a transcriptional activator of progesterone-responsive genes, recent studies have identified a distinct region of the PR-A that is associated with a trans-dominant repressor function of other steroid hormone receptor genes [11, 12]. In addition, different ligand binding capacities between PR isoforms at low receptor concentrations have been postulated [13], and furthermore, these isoforms can positively influence each other [14]. In vitro studies suggest that human PR-A and PR-B have specific characteristics and that their function can be regulated at different levels [15, 16]. Immunohistochemistry techniques have been used to localize ovarian PRs [17, 18]; but to distinguish between the different mRNA isoforms and to quantify the levels of PR isoforms, other techniques are required. The presence of PR-A and PR-B in the monkey and human CL has recently been identified [19, 20]; however, the distribution and regulation of PRs in the human CL during its life span in the menstrual cycle have not been fully characterized. In the present study, we surveyed the expression pattern of PR mRNA isoforms B and A/B and the cellular distribution of PR-A/B mRNA. We also determined the PR-A/B protein levels and cellular localization during different phases in the human CL in relation to luteal tissue concentrations as well as serum levels of progesterone. In order to investigate further the regulatory role of PRs in human CL function, functional studies were undertaken of PR-A/B blockage following LH–receptor stimulation in an in vitro model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients

Altogether twenty-nine women were recruited. All had given informed consent, and the study was approved by the Ethical Committee of Umeå University Hospital (Umeå, Sweden). Ovarian tissue was obtained upon commencement of elective surgery for benign conditions (i.e., legal sterilization, n = 13, or uterine fibroma, n = 16) at the Department of Obstetrics and Gynecology, Umeå University Hospital. The patients had not received any hormonal therapy during the preceding month and were otherwise healthy. The average age of the patients was 39.6 yr (range 29–47). All patients had proven fertility and had a history of regular menstrual cycles ranging between 24 and 30 days. The CL age was determined according to onset of the last period of menstruation and detection of an ovulatory LH surge in urine (Clearplan One Step; Unipath Ltd., Bedford, UK). Day 1 (ovulatory day) was defined as the first day after a positive LH test. According to these two parameters, the patients were scheduled for investigation in early (Day 2–5), mid (Day 6–10), or late (Day 11–14) LP. On the day of surgery, a preoperative ultrasound was performed to localize the CL, and blood samples were taken. Immediately after abdominal access, the CL was extirpated, divided into pieces, and placed in liquid nitrogen or prepared for immunohistochemistry by immersion fixation. Some CLs were used for cell cultivation (n = 3) and were immediately transported on ice to the laboratory. To further characterize the patients and exclude possible cases of CL insufficiency, serum levels of steroids were measured. As a consequence of those results, two patients were excluded because of low serum progesterone levels and ultrasonographic characteristics suggestive of insufficient CL function [21].

RNA Preparation

Total RNA was isolated using CsCl-gradient ultra-centrifugation and phenol/chloroform extraction [22, 23]. The RNA concentration was determined by spectrophotometry, and the integrity of total RNA samples was verified by 1% agarose gel electrophoresis in the presence of ethidium bromide followed by visualization under UV light.

Semiquantitative Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Method 1

A previously validated RT-PCR methodology, essentially as described before [24, 25], was employed to determine PR gene expression in isolated CL tissue. For all the samples from each experiment, cDNA was synthesized from 2.0 µg total RNA, using a NotI-d(T)18 primer (Ready-to-go; Pharmacia, Uppsala, Sweden). After incubation at 37°C for 1 h, the reaction was quenched by heating to 95°C for 5 min and stored at -20°C until use in PCR. A 1.0-µl aliquot of the cDNA reaction mixture was used for 27–29 cycles (Table 1) of PCR amplification (30 sec at 94°C, 30 sec at 55°C, 90 sec at 68°C, and after the final cycle, a 5-min 68°C extension step). PCR reactions were carried out in the presence of 1.75 mM MgCl2, 50 mM Tris HCl (pH 9.2), 16 Mm (NH4)2SO4, 350 µM dNTP, and 1 U Taq polymerase (Boehringer Mannheim, Mannheim, Germany) for each 20-µl reaction. To further validate the true identity and to enhance quantification range, an aliquot of PCR products was electrophoresed through 1% agarose gels, transferred to nylon membranes (Hybond N+; Amersham, Oakville, ON, Canada), and subjected to standard Southern hybridization. Specific internal cDNA probes were PCR-amplified, cloned into plasmid vectors (pCRII-TOPO TA cloning kit; Invitrogen, Carlsbad, CA), sequenced, and labeled by the PCR fluorescence labeling mix (Boehringer Mannheim; Table 1). Hybridizations were carried out overnight at 60°C and were followed by several washes, of which the final wash was 0.1-strength SSC (single-strength SSC is 0.15 M saline and 0.015 M sodium citrate) and 0.1% SDS at 60°C. Membranes were exposed to x-ray films (Hyperfilm MP; Amersham, Buckinghamshire, UK) between intensifying screens for 30 sec to 5 min. PCR amplification controls were also performed by amplification of products of similar sizes of glyceraldehyde-3-phosphate dehydrogenase (G3PDH, primer sequence I, Table 1) from the same cDNA target, as an internal control. The autoradiograms were scanned for intensity of hybridization using a computerized visual light densitometer (Fast Scan-Computing Densitometer series 300; Molecular Dynamics, Baltimore, MD), and arbitrary densitometric levels of respective signals were determined. In all experiments, negative controls were included (without reverse transcriptase in the RT reaction or without cDNA in the PCR reactions), thereby ruling out cross contamination between samples. The possibility of genomic DNA amplification was further excluded by designing intron-spanning primers. When both gene products were amplified from pooled cDNAs obtained from uterine endometrial cells with incremental amounts of total RNA subjected to RT and subsequently co-electrophoresed through an agarose gel, PCR amplification of PR mRNA yielded linear increases up to 35 cycles (data not shown).


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  		TABLE 1. Position and sequence of synthesized oligonucleotides, number of cycles used for PCR amplifications of G3PDH (Genbank accession number x01677) and PR-A/B and -B cDNAs (Genbank accession number M15716), and position sequence data of internal control probes used for Southern blotting

Semiquantitative RT-PCR Method 2

To further substantiate results, a different approach recently described in detail [26] was also used. Briefly, the concentration of plasmid DNA, with cloned inserts of the respective gene segment of interest, was dissolved in distilled water and quantified by spectrophotometry followed by calculation of the number of copies of each plasmid DNA. Then the solutions of plasmid DNA at known concentrations (104-106 copies/µl) were prepared as standard templates. These standard templates and cDNAs from the unknown samples were amplified simultaneously using the same master-mix solution. PCR was carried out in the presence of a 1-µg sample of cDNA or of a concentration of each standard plasmid DNA, using the same conditions as mentioned above (method 1). Each product was electrophoresed on a 1% agarose gel, stained with ethidium-bromide, visualized under UV light, and photographed with negative film (T667, ISO3000; Polaroid, Hertfordshine, UK). The films were scanned for measurements of integrated optical density, and the arbitrary densitometric level of respective signals was determined. A standard sigmoid curve for each gene was drawn; then the copy number in the sample was calculated and quantified from the respective standard curve. The relative mRNA amounts of PR-B and PR-A/B versus G3PDH (primer sequences G3PDH II, Table 1) were calculated by dividing each PR-B and PR-A/B copy (copy/1 µg cDNA) by the corresponding G3PDH copy (copy/1 µg cDNA).

PR Protein Determinations

Detection of total cellular PR-A/B protein was determined by an enzyme immunoassay (Abbott Laboratories, Abbott Park, IL). The amount of receptor protein was correlated to the content of DNA in the CL specimen, as analyzed using the diphenylamine method [27]. Receptor concentration was expressed as femtomole of receptor per microgram of DNA, and a value equal to or higher than 0.1 fmol PR/µg DNA was considered receptor-positive.

Immunohistochemistry

CL tissues were immersion-fixed in Bouin's solution for 4 h, dehydrated, and embedded in paraffin. Sections (4-µm thick) were cut and stained with hematoxylin and eosin. Adjacent 4-µm-thick sections were deparaffinated, rehydrated, and retreated with 3% H2O2 to suppress endogenous peroxidase activity; washed with PBS (pH 7.2); and heated in a microwave oven (600 W) for 2 x 7.5 min in citrate buffer (0.01 M, pH 6.0) as earlier described [28]. Thereafter, slides were incubated overnight with a primary monoclonal antibody (26 mg/L) (Dako Ltd., Glostrup, Denmark) raised against amino acids 533–547 of the human PR-A/B protein [29]. The sections were then incubated in chronological order with a biotinylated secondary goat anti-rabbit antibody for 30 min, with avidin-biotin-peroxidase complex (ABC) reagents for 45 min, and with peroxidase substrate (AEC: 3-amino-9-ethyl carbazole; Vector Laboratories, Burlingame, CA) for development for 15 min. Between incubations, the sections were washed for 10 min in PBS solution. Before examination, the sections were lightly counterstained with Mayer's hematoxylin solution. For positive controls, sections from urinary bladder were used. Immunoreactivity was thereafter evaluated in a light microscope at x200 magnification.

In Situ Hybridization

To localize the expression of progesterone type A/B receptor mRNA in the human CL, a 231-base pair (bp) cRNA probe (Table 1) was synthesized using a DigRNA labeling kit (Promega, Madison, WI). The in situ hybridization was performed essentially as previously described [30]. Briefly, 10-mM cryostat sections were collected on SuperFrost/Plus slides (Menzel-Gläzer, Brann Scheig, Germany) and fixed in 4% paraformaldehyde (in PBS) for 10 min. Prehybridization was performed in a solution of 50% formamide, 5-strength SSC, 5-strength Denhardt's solution, 250 mg/ml tRNA, and 500 mg/ml herring sperm DNA at room temperature overnight. Hybridization was performed in the same solution containing approximately 1 µg/ml digoxigenin-labeled (Boehringer) riboprobe at 72°C overnight. After hybridization, the slides were washed in 0.2-strength SSC at 72°C for 1 h. The slides were then incubated with blocking buffer (10% heat-inactivated normal goat serum, 0.1 M Tris pH 7.5, 0.15 M NaCl) for 1 h at room temperature before addition of the alkaline phosphatase-conjugated antidigoxigenin antibody (1:5000 dilution in 0.1 M Tris pH 7.5, 0.15 M NaCl, 1% heat-inactivated goat serum). After incubation with the antibody at 4°C overnight, the slides were washed 3 times in Tris buffer (0.1 M Tris pH 7.5, 0.15 M NaCl) before being equilibrated in alkaline phosphatase buffer (0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl2). The substrate for alkaline phosphatase (450 µg/ml 4-nitro blue tetrazolium chloride and 175 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate in alkaline phosphatase buffer) was then added. After between 6 h and 3 days in the dark, the color reaction was terminated with 10 mM Tris and 1 mM EDTA (pH 8.0), results were evaluated by light microscopy, and slides were documented by photography.

Luteal Cell Culture Procedure

All cell culture reagents and drugs were purchased from Life Technologies/Gibco BRL (Gaithersburg, MD) unless otherwise specified. Freshly obtained CL tissue was immediately transported to the laboratory in ice-chilled incubation medium, where the tissue was carefully minced and enzymatically dissociated in sterile filtered M199 containing 1.0 mg/ml collagenase type V, 50 µg/ml deoxyribonuclease 1, 1.5% BSA, and 0.95 mM CaCl2 (all from Sigma-Aldrich Corporation, St. Louis, MO). The cell suspension combined with an equal volume of sterile saline (0.154 M) was layered onto 3.0 ml of a fixed Percoll gradient (density 1.117 g/ml) and centrifuged at 400 x g for 40 min to remove blood cells and cellular debris. The enriched luteal cells were carefully collected from the interface, washed, and resuspended in fresh M199 containing 26 mM NaHCO3, 25 mM Hepes, 50 U/ml penicillin, 50 µg/ml streptomycin, and 1% heat-inactivated fetal bovine serum. Cells were counted in a Bürker chamber under a light microscope, and the viability in all experiments was estimated to be above 90% by the trypan blue dye exclusion method. The volume of the cell suspension was adjusted with M199 to give a concentration of 1.5 x 105 cells/ml medium, and the cell suspension was added to cell culture dishes (Nunclon, Nunc A/S Roskilde, Denmark) and preincubated at 37°C in humidified air/5% CO2 for 18–24 h in a Forma-Scientific CO2 incubator, model 3196 (Marietta, OH). After a change to fresh medium, adherent cells were treated with incremental doses of hCG (Profasi; Ares-Serono S.A., Geneva, Switzerland) or the PR antagonist mifepristone (RU-486; Exelgyn, Paris, France), in triplicate wells. The concentration used in the dose response test for mifepristone ranged from 1 nM to 10 µM and for hCG from 0.001 U/ml to 1.0 U/ml. To study the integrated response to agonists used, a high dose of hCG (0.1 U/ml) was chosen and added to all wells while increasing doses of mifepristone (range 1 nM to 10 µM) were added. Cultures were terminated after 24 h, and the medium was collected and stored at -20°C until assayed for progesterone concentration.

Hormonal Assays

Pieces of CL tissue were weighed (33.6 ± 3.3 mg, n = 26) and placed in 95% v:v ethanol for 48 h at 4°C in thoroughly sealed tubes according to procedures previously described in detail [31]. Concentrations of progesterone in CL tissue, serum, or cell culture medium were determined by a time-resolved fluorometric assay (DELFIA; Wallac Ltd., Turkku, Finland) according to the manufacturer's protocol. All samples were analyzed in duplicate in one assay, in which the coefficient of variation was < 5%.

Statistical Analysis

Values are given as mean ± SEM or as individual values. Differences between groups were tested using the nonparametric Mann-Whitney U test. Pearson's correlation test and linear regression were used when appropriate. Each cell culture experiment was repeated three times; the progesterone data are from triplicate determinations and were pooled from all patients after normalization of control values to 100%; they are presented as mean ± SEM. In cases of combined treatment with hCG and mifepristone, the value of hCG treatment alone was standardized to 100%. A P value below 0.05 was regarded as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The patterns of CL tissue and peripheral serum progesterone concentrations during the three different postovulatory CL developmental stages are presented in Figure 1. In relation to CL age, changes in tissue levels of progesterone were somewhat different from those in serum, since highest tissue concentrations were found during early and mid LP, whereas peripheral serum progesterone levels were significantly lower in early LP than peak levels obtained at mid LP (Fig. 1). In late LP, progesterone concentrations in both peripheral serum and CL tissue were significantly depressed.



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  		FIG. 1. Progesterone concentrations in serum and CL tissue, detected by time-resolved fluorometric assay (DELFIA) during early (Days 2–5), mid (Days 6–10), or late (Days 11–14) LP. P values from Mann-Whitney U test analysis of differences between groups are depicted

After positional cloning of a human ovarian cDNA coding for the major part of hormone- and DNA-binding domains of the PR gene, methods for quantifying PR mRNA isoforms in the human CL were developed. Sequence analysis revealed that the obtained cDNA was > 99% homologous to the previously reported sequence [10]. Two methods were employed to detect changes in PR mRNA transcript abundance, both based on the ability of RT-PCR to amplify to quantifiable amounts of DNA in proportion to the copy numbers of template cDNA (or mRNA). Both methods gave similar results, and data presented are all from method 2. On the basis of calculations in relation to constitutively expressed G3PDH (the levels of which we have previously demonstrated do not change throughout the luteal phase, [32]), PR-B mRNA was considerably less abundant (approximately 100- to 1000-fold) than PR-A/B (Fig. 2A). When the PR-A/B receptor mRNA levels were grouped according to the age of the CL (Fig. 2B), or when they were individually expressed as percentage change of the Day 3 value, no statistically different amounts of steady-state levels of PR-A/B receptor mRNA levels or any statistical correlation (r2 = 0.026, P = 0.48, Pearson's correlation test) with CL age in days was found. Contrasting to this pattern was the finding that PR-B mRNA levels were significantly reduced (-46%) in mid LP compared to the late LP (Fig. 2C). The relative amounts of PR isoform mRNA, expressed as the ratio of PR-B over PR-A/B mRNA, were calculated to be decreased by 35% in mid LP compared to early LP (P < 0.05, n = 24).



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  		FIG. 2. Quantification of G3PDH, PR-A/B, and PR-B mRNA by RT-PCR as described in Materials and Methods. Plasmid DNA containing the respective gene fragment at known concentration and cDNAs from the unknown sample were amplified simultaneously using the same mastermix PCR solution. After electrophoresis, Southern transfer, hybridization with internal cDNA probes, and measurements of the integrated density of each band, the copy number was calculated from the standard curve (A). B) Ratio of progesterone receptor A/B mRNA and G3PDH mRNA expression and C) ratio of progesterone receptor B mRNA and G3PDH mRNA expression during different age-periods of the human CL. Differences between groups was tested by the Mann-Whitney U test

In whole CL tissue, the PR-A/B receptor concentrations, as measured by an enzyme immunoassay, were moderately high and unchanged in early and mid LP, but were significantly reduced by 48% in the late LP (Fig. 3A). This decline of PR-A/B abundance was similar to changes in CL tissue concentrations of progesterone (Fig. 1). Interestingly, PR-A/B receptor concentrations were significantly and inversely correlated to the age of the CL (Fig. 3B). Immunohistochemical staining for PR-A/B antigen and in situ hybridization of PR-A/B mRNA were used to more precisely localize PR-expressing cells in the CL. As can be seen in Figures 4 and 5, the regional distribution pattern of the staining was distinctly localized to the luteal cells in the steroidogenic layer, while only background levels were seen in connective tissue, vascular endothelium, and internal parts of the CL.



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  		FIG. 3. Enzyme-linked immunoadsorbent assay of total cellular PR-A/B protein during different periods (differences between groups tested by Mann-Whitney U test) (A) and different days (B) of the luteal phase (linear regression employed, dotted lines indicates 95% confidence intervals, r2 = 0.34, P < 0.01, n = 23)



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  		FIG. 4. Localization of PR-A/B mRNA in the mid-luteal CL. Cryostat sections (10 µm) were hybridized with a digoxigenin-UTP-labeled progesterone receptor antisense RNA probe (A) and a sense RNA probe (B). Specific hybridization was localized to the steroidogenic cells of the granulosa-lutein compartment, while only background levels were seen in the theca-lutein compartment. x100 (published at 58%)

The functional role of progesterone receptor-mediated action was evaluated by use of a progesterone receptor antagonist, using an earlier established in vitro culture system of enriched luteal cells obtained from freshly excised mid-LP CL [32]. Under the conditions used herein, the cell viability, assessed by trypan blue exclusion, exceeded 90%. As expected, after a 24-h preincubation period, these cells responded to treatment with hCG with up to 2-fold increases in progesterone output (Fig. 6A). Under these circumstances, no clear and statistically significant response to mifepristone treatment alone in luteal cell progesterone synthesis was noted (Fig. 6A). However, when the integrated response to hCG stimulation and progesterone receptor blockage was tested, a dose-related inhibition of progesterone synthesis was found (Fig. 6B). A low dose of mifepristone (10 nM) was found to be the least amount needed to significantly reduce progesterone synthesis, whereas maximum inhibition (-47%) was seen at 10 mM (Fig. 6B).



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  		FIG. 6. Progesterone synthesis by cultured enriched human luteal cells obtained on Days 6–9 (mid LP) after the LH rise (see Materials and Methods). Values are standardized to 100%, according to the value obtained from untreated cells. Triplicate wells containing 1.5 x 105 cells/ml were treated with either 0.1 U/ml hCG or incremental doses of mifepristone alone (A). In B, cells were treated with 0.1 U/ml hCG in addition to incremental doses of mifepristone (RU-486). *P < 0.05 versus control, n = 3. Differences between groups was tested by the Mann-Whitney U test


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We describe herein the identification, isolation, regional distribution, and regulation of PR coding mRNAs and PR A/B protein, in addition to a putative functional role of PRs as steroid synthesis modulators in the steroidogenic cells of the human CL. Tissue was obtained from prospectively recruited, regularly cycling, fertile, hormonally untreated, and otherwise healthy women; and a high precision in establishing the actual CL age was secured by relating the postovulatory period to number of days following the LH surge, rather than by endometrial histology [33]. The pattern of systemic concentrations of progesterone compared to intraluteal cellular progesterone concentrations was monitored throughout the secretory phase of the menstrual cycle, and our results suggest that serum progesterone levels do not directly reflect the elevated progesterone amounts in the younger CLs. Whether this is caused by increased clearance/metabolism or a secretory capacity that is not fully developed (possibly because of CL blood vessels that are not yet fully developed) remains unexplained, but similar findings have been reported in the monkey CL [34].

Data presented herein demonstrate unequivocally that the progesterone target cells have the same regional localization and distribution as the previously characterized steroidogenic cells, which are sensitive to extrinsic gonadotropic control by LH and hCG [30, 32]. Previous studies have described a cellular distribution pattern of PR immunoreactivity both in the nonhuman primate CL [35, 36] and the human CL [17, 18, 37], with findings similar to ours. However, the present study is, to the best of our knowledge, the first report in which in situ hybridization has been used to detect PR mRNA in the primate CL. This mRNA is expressed in relatively high abundance in the luteal cells comprising the steroidogenic layer, whereas only background levels were seen in connective tissue, vascular endothelium, and internal parts of the CL.

In the search for a putative transcriptional regulation of PR gene expression in the human CL, quantitative analysis of mRNA and protein concentrations were performed, and comparisons were made based on three arbitrarily established postovulatory age groups representing the CL developmental stages. Our data suggest that the ratio of PR mRNA isoforms is altered during these different developmental stages of the CL and that the total luteal PR-A/B receptor concentration is inversely correlated to CL age. This is suggestive of a relation to luteal progesterone synthesis, since PR-A/B levels closely follow intraluteal progesterone concentrations, a finding that in turn is consistent with the earlier hypothesis of a functional dependence on progesterone action within the CL [38]. Previous immunocytochemical studies in the monkey [35] and human [17, 18] have indicated that the percentage of PR-positive cells was low in the early LP, peaked at mid LP, and was very low by late LP. Since our immunocytochemical method does not allow for direct quantitative comparisons, we employed an enzyme immunoassay widely used in clinical laboratories to measure PR-A/B protein concentrations [39]. With the exception of a moderate to high expression in earlier luteal stages, the finding herein of decreased PR protein amounts in later stages of CL development is in good agreement with these earlier reports [17, 18, 35]. With the exception of the recently published study by Misao et al. [20], to the best of our knowledge no other studies have attempted quantification of both PR-A/B and PR-B mRNA levels in the human CL. During mid LP (which is the period of anticipated implantation, provided that the cycle resulted in a fertilized ovum), the mRNA levels coding for PR-A/B were maintained at an unaltered level while PR-B mRNAs were markedly down-regulated. Whether this is also reflected in decreased amounts of functional PR-B protein in the CL remains to be determined. A previous study has described different ratios of PR-A to PR-B levels in various tissues from the monkey reproductive tract, including the CL [19]. However, the finding that PR-B levels are higher than PR-A levels in the monkey CL is not in accordance with recently reported findings in the human CL [20]. Furthermore, results presented herein suggest a 100- to 1000-fold lower abundance of PR-B mRNA than of PR-A/B mRNA. Whether these differences are related to interspecies differences or are due to different methodologies remains unsettled, and as such provides a basis for additional studies.

In both rhesus monkeys [40] and baboons [36], CL PR-A/B mRNAs reportedly increase with the age of the CL, whereas low levels of immunoreactive PR have been detected despite elevated mRNA levels [19, 36]. These divergences in concentrations have previously been interpreted to be caused by different methods of quantitating mRNA or by posttranscriptional stabilization or translational regulation. On the basis of the available evidence, it is likely that, in addition, interspecies differences between the monkey and human CL exist. Furthermore, it should be recognized in this context that CL tissue prepared for Northern blot, Western blot, or RT-PCR contains many different cell types [1], and uncharacterized differences in the cellular population of specimens studied may account for these discrepancies. Ideally, while the possible drawbacks of architectural and functional disorganization must be recognized, individually typed and purified luteal cells could provide a better study object. However, results of such protocols remain to be seen.

Most of the available evidence describing mechanisms by which progesterone may influence progesterone synthesis and structural integrity of the CL has been generated from subprimate and nonhuman primate animal models. Limited data are available from human ovarian cells indicating that treatment with mifepristone (RU-486) will dose-dependently inhibit 3-ß-hydroxysteroid dehydrogenase and 17{alpha}-hydroxylase activities in cultured human granulosa-lutein cells [41, 42]. Progesterone is also proposed to inhibit granulosa cell proliferation and promote differentiation into a functional luteal cell phenotype, and thereby increase steroidogenic potential [5]. In addition to endocrine control by LH or hCG, several locally produced agents (for references, see [1, 3]) have been postulated to positively influence progesterone synthesis (i.e., insulin-like growth factor I) [43] or to act in an inhibitory (luteolytic) fashion (i.e., prostaglandin F2{alpha}) [32]. While experiments presented herein were limited only to mid LP, the results suggest a strong dose-related inhibitory action on hCG-induced progesterone synthesis. Taken together, they appear to validate the assumption that the very high concentrations of progesterone found within the CL (range 0.31–7.50 µM/mg wet weight) may also exert effects mediated via an auto- and/or paracrine loop through PRs, the activation of which in turn are intimately involved in the functional regulation of the human CL.

In summary, PR mRNA and PR protein were detected in steroidogenic cells of the human CL throughout the postovulatory period of the menstrual cycle. The change in ratio of expression of PR isoforms mid-luteally, favoring type A/B, and the decrease of hCG-induced progesterone synthesis by mifepristone, suggest an important regulatory role of progesterone in the steroidogenic cells of the CL. The recent demonstration of a membrane-bound progesterone-binding site in the bovine CL [44], in addition to the existence of a truncated third receptor isoform, termed PR-C [45], implies that a compound model of progesterone action in ovarian target cells operates at a highly complex level. The exact molecular mechanism(s) remain to be deciphered in future experiments.



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  		FIG. 5. Immunohistochemical detection of PR-A/B protein in the human CL (arrows) in a Day 3 CL (x200; A) and a Day 7 CL (x200; B) (published at 52%). Arrows indicate specific nuclear staining of both large and small steroidogenic luteal cells. No staining was seen in vascular endothelium


   ACKNOWLEDGMENTS
 
The skillful technical assistance by Ms. Monica Isaksson and Ms. Birgitta Ekblom is warmly appreciated.


   FOOTNOTES
 
First decision: 5 August 1999.

1 Supported by grants from The Swedish Medical Research Council K99-72X-13144-01A and K98-17P-11832-03C (J.I.O.) and by the Cancer Research Foundation of the Department of Oncology at Umeå University, The Swedish Society of Medicine, and The Swedish Society for Medical Research. Back

2 Correspondence. FAX: 46 90 773905; jan.olofsson{at}obstgyn.umu.se Back

Accepted: October 22, 1999.

Received: June 2, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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