Molecular Human Reproduction, Vol. 5, No. 8, 714-719,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Cell characteristics and function of two enriched fraction of human luteal cells prolonged culture
1 Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden, 2 Department of Women's and Children's Health, Uppsala University, Uppsala University Hospital, Uppsala, Sweden, and 3 Department of Clinical Science, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden
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Abstract |
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Two subpopulations of steroidogenic cells exist in the corpus luteum of most species. The aims of the present study were to characterize these cells and to study their function during long-term culture. Human corpora lutea from early and late luteal phases were treated by mechanical and enzymatic digestion, followed by density sedimentation. Five distinct cell bands were obtained, two of which produced large amounts of progesterone. These were characterized according to density, size, steroidogenic enzymes, and numbers. More than 75% of cells expressed immunoreactive 3ß-hydroxydehydrogenase (3ß-HSD). Cells of higher density/smaller size were obtained in increasing numbers during the luteal phase and were more numerous compared with large cells. Under basal, human chorionic gonadotrophin (HCG)-, and prostaglandin E2-stimulated culture conditions, progesterone synthesis was greater in large cells of the early, but not late, luteal phase. Both cell fractions obtained from late, in contrast to early, luteal phase increased their basal progesterone production during the culture period of 9 days. We conclude that this technique for luteal cell isolation in the human yields two distinct subpopulations of steroidogenic cells, which respond differently to luteotrophic stimuli. We also conclude that cells of late luteal phase readily increase their progesterone synthesis over a period of 9 days, indicating a transition to longevity.
corpus luteum/human/progesterone
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Introduction |
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The regularity of the menstrual cycle, as well as the maintenance of early pregnancy, are dependent upon a normal corpus luteum (CL) function. The CL of most species consists of two types of steroidogenic cells distinguished by certain morphological and functional characteristics (Fitz et al., 1982
; Rodgers and O'Shea, 1982; Rodgers et al., 1983; Ohara et al., 1987; Nelson et al., 1992; Retamales et al., 1994). The most apparent morphological difference between the two types of luteal cells is their size; consequently they are generally referred to as large and small luteal cells. In domestic animals, such as the cow and sheep, both luteal cell types synthesize significant amounts of progesterone when cultured in vitro (Fitz et al., 1982; Rodgers and O'Shea, 1982; Rodgers et al., 1983; Hoyer et al., 1984; Niswender et al., 1985; Lei et al., 1991). The basal production of progesterone is generally greater in large luteal cells compared with small luteal cells (Rodgers et al., 1983) and this is reflected in their ultrastructure. Small cells, however, usually show a proportionally greater increase in progesterone production, when exposed to human chorionic gonadotrophin (HCG), suggesting different mechanisms in regulating steroidogenesis (Fitz et al., 1982; Rodgers and O'Shea, 1982; Rodgers et al., 1983; Ohara et al., 1987; Retamales et al., 1994). Decreasing progesterone concentrations in peripheral blood is one of the first indicators of luteolysis. This initial functional luteolytic stage seems to be reversible, since luteotropic agents, i.e. HCG and prostaglandin E2 (PGE2), reverse early stage luteolysis in human luteal cells in vitro (Dennefors et al., 1982; Fitz et al., 1984; Hahlin et al., 1988; Hagström et al., 1996). It is, however, not fully understood if luteolysis primarily depends upon direct action of luteolytic factors, a decline of luteotrophic stimuli, or a combination of these mechanisms.
Most experimental data concerning the function of the two subpopulations of luteal cells and their different morphological and functional properties have been obtained from experiments with CL cells from domestic animals (Fitz et al., 1982; Rodgers et al., 1983; Niswender et al., 1985). Few attempts have been made to study these properties in human luteal cells (Ohara et al., 1987; Retamales et al., 1994; Carrasco et al., 1996) and all have involved short-term cultures. In order to study changes in the cells that may be responsible for initiating luteolysis, cultures for longer periods of time are necessary. The aims of this study were three-fold: (i) to design an experimental model for such prolonged culture of the enriched fractions of the two subclasses of luteal steroid-producing cells in the human; (ii) to characterize these cells concerning density, size, and steroidogenic properties in relation to luteal phase; and (iii) to examine the steroidogenic capacity of the cells during prolonged culture for periods possibly beyond their predestined life-span during a non-conception cycle with and without luteotrophic influence.
Materials and methods
Subjects
Corpora lutea (n = 19) were obtained from women aged 25–45 years undergoing abdominal surgery for a variety of benign non-ovarian conditions, typically myoma uteri. All women gave informed consent prior to the study, which was approved of by the local Ethics Committee at Göteborg University, Sweden.
Dating of corpus luteum
Detailed records were taken regarding the pattern of the three previous menstrual cycles, the date of last menstrual period and symptoms of ovulation. Only women with regular menstrual cycles (cycle length 25–33 days) were included in the study. The women had been without hormonal treatment for at least 3 months prior to surgery. Evaluation of the gross morphology of the CL whilst in situ, as well as immediately after excision, was undertaken to examine whether the morphological appearance of the CL matched the date of the cycle. Based on the information compiled, the CL were classified as either early luteal phase (0–4 days after ovulation, n = 7) or late luteal phase (>9 days after ovulation, n = 9). Three CL used for the initial cell suspension experiments were not dated. In each experimental group, the number of CL vary depending on variations in cell yield at each preparation.
Cell preparation
All procedures were performed under sterile conditions. The CL was removed in toto, leaving the ovary, at the beginning of the surgical procedure and immediately placed into ice-cold Ca2+ and Mg2+-free phosphate-buffered saline (PBS; Life Technologies Inc, Paisley, Scotland, UK). Within 30 min, the capsule and large blood vessels were removed, followed by separation of the central blood clot. The remaining tissue was then cut into small pieces (wet weight ~1 mg), placed into 10 ml of PBS supplemented with collagenase CLS 2 (2.5 mg/ml; Worthington Biochemical Corporation, Freehold, NJ, USA) and DNAse type 2 (50 µg/ml; Boehringer-Mannheim, Mannheim, Germany), and incubated in a shaking water bath (37°C) for 60 min. The digested tissue specimens were then strained through a 100 µm mesh filter (Falcon®; Becton Dickinson, Franklin Lakes, NJ, USA), pelleted at 200 g for 5 min, washed twice in 10 ml of PBS, and once again centrifuged at 200 g for 5 min. Finally, the cell pellet was resuspended in 1 ml PBS and layered on a Percoll® (Pharmacia-Upjohn, Uppsala, Sweden) discontinous gradient of 63, 54, 45, 27, and 18% layers (densities = 1.088, 1.076, 1.064, 1.041, and 1.029 g/ml respectively) and centrifuged for 20 min at 400 g. Cells were recovered from all four interfaces, washed three times with cell culture medium (minimal essential medium with Earle's salts; Life Technologies Inc, Paisley, Scotland, UK) supplemented with 292 mg/ml L-glutamine, 10% fetal calf serum, and 50 µg/ml gentamicin. Samples from each band were counted in a haemocytometer and assessed by the Trypan Blue exclusion method. Cell viability was >90% in all experiments.
Characterization of cell populations
In initial experiments, all five cell bands were cultured for 48 h after which progesterone concentrations in conditioned media were analysed. The two cell bands producing progesterone were further analysed by flow cytometry. 1.5x106 cells from each of the steroid producing cell bands were resuspended in 1 ml of PBS. The cells were immediately run through a flow cytometer (Cytoron Absolute; Ortho, Raritan, NJ, USA) and counted based on their cell size in forward scatter. In principle the laser beam is scattered each time it hits a cell. The larger the area of the cell, i.e. size, the larger proportion of the laser beam is scattered. Size groups of cells produced scatter peaks in arbitrary units.
In one additional experiment on a CL of late luteal phase the two cell bands producing progesterone were also analysed concerning expression of 3ß-hydroxydehydrogenase (3ß-HSD) by means of immunohistochemistry. The cells were cultured on chamberslides and were treated with 0.3% (w/v) H2O2 in methanol for 30 min (to minimize endogenous peroxidase activity), incubated overnight (4°C) with rabbit polyclonal anti 3ß-HSD antibodies (1:400) (generously provided by Dr I.Mason, Edinburgh, Scotland, UK) followed by three washes of PBS. A sensitive detection kit (Vecta Stain ABC kit) with a secondary horse anti-rabbit antibody was used. The slides were counterstained in haematoxylin and mounted in Pertex (Histolab, Göteborg, Sweden). Positive staining was visualized as brownish reaction product.
Experimental procedures and assays
Luteal cells (75x103 cells in 0.5 ml culture medium per well) were seeded into 24-well tissue plates, and cultured at 37°C in an atmosphere of 5% CO2 in air. The cells were allowed to attach for 24 h. Experiments started with the first change of culture medium (0.5 ml). During the next 48 h the cells were cultured, with or without exogenous HCG (10–1000 IU/l; Profasi, Serono, Rome, Italy) or PGE2 (1–100 µg/l; Sigma, St Louis, MO, USA). During prolonged culture, media with or without additives were changed at 48 h intervals. The culture medium was stored at –20°C until analysed. Progesterone was analysed by a time-resolved fluoroimmunoassay (Delfia; Wallac Oy, Turku, Finland) with inter- and intra-assay coefficients of variation of <9.6%. The protein content in the wells was determined by the Pierce BCA Protein Assay (Pierce, Rockford, IL, USA).
Statistical analysis
Each individual datapoint represents the mean of two or three wells. The absolute values are expressed as the mean and range. In order to perform statistical analysis the numbers have been log transformed, thereby achieving a normalization of the material and an acceptable skewness and curtosis. With log-transformed numbers, statistical analysis was performed by 1x2 analysis of variance. P < 0.05 was considered to be statistically significant. Numbers are given as means ± SEM.
Results
Cell populations in corpora lutea of early and late luteal phase
Five distinct bands of cells were identified in the Percoll gradient. The progesterone concentrations in media and the protein content following culture for 48 h of each band are depicted in Table I. Further investigations and functional experiments were carried out on the two cell bands with apparent capacity to produce progesterone. These cells were retrieved from the bands at the 18/27% and 27/45% interfaces, being cells of lower density (1.041–1.029 g/ml) and higher density (1.064–1.041 g/ml) respectively. Immunohistochemical staining with antibodies against 3ß-HSD revealed that >75% of cells in each fraction were positive. By means of flow cytometry, the size of the lower density cells (peak at forward scatter around 96 arbitrary units) was determined to be larger than the size of the higher density cells (peak around 84 arbitrary units). These cells are hereafter referred to as large CL cells (LCLC) and small CL cells (SCLC) respectively. Significantly (P = 0.035) more SCLC than LCLC were obtained after preparation in the late luteal phase, while no significant difference was noted for the early luteal phase. SCLC were generally more numerous than LCLC. The numbers of retrieved SCLC increased from CL of early (SCLC/early: 5.6x106 cells, range 2.2–9.9) compared with late (SCLC/late: 7.6x106 cells, range 4.4–10.3) luteal phase whereas the number of LCLC decreased (LCLC/early: 4.2x106 cells, range 1.1–11.3; LCLC/late: 1.8x106 cells, range 1.2–2.8) during the course of the luteal phase.
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Progesterone production of enriched luteal cells in short-term culture
During a 48 h culture period, both luteal cell types responded to HCG with a dose-dependent increase in progesterone synthesis (Figure 1
). In CL from early luteal phase, the progesterone production was higher with a mean basal progesterone formation during a 48 h culture period of 92.1 nmol/l for LCLC and 37.2 nmol/l for SCLC. In CL from late luteal phase, the mean basal progesterone formation was 6.7 nmol/l for LCLC and 18.3 nmol/l for SCLC. LCLC from the early luteal phase synthesized higher concentrations of progesterone than SCLC. No difference was noted between the cell types during the late luteal phase. The progesterone synthesis from cells stimulated by HCG was significantly greater (P < 0.0001 for all groups) for concentrations 100 IU/l and 1000 IU/l compared with controls, but the relative stimulatory effect of HCG was more pronounced in cells obtained from the late luteal phase than the early luteal phase (Figure 1), and reached saturation at the highest concentration. The significant difference between controls and 10 IU/l HCG stimulation was significant only for the cells from late luteal phase (P = 0.11 and P = 0.0326 for early and late phase cells respectively). Figure 2 depicts the dose-dependent response in progesterone synthesis to PGE2. No significant difference was noted between fractions of cells. Contrary to the phase dependent response to HCG, no difference in response to PGE2 could be noted in cells from early or late luteal phase.
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Prolonged cell culture
Basal progesterone synthesis during prolonged culture of luteal cells of different age and size is depicted in Figure 3
. CL cells in the early luteal phase demonstrated, regardless of size, a continuous decline in progesterone production during prolonged culture. In contrast, the cells of the late luteal phase demonstrated a continuous increase in progesterone synthesis, and this was especially pronounced in SCLC. The difference between cells of early and late luteal phase was significant (P = 0.046).
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In separate experiments, the protein content was measured at 1, 3, 5, 7, 9 days of culture. The protein content increased in cultures of both cell types from early and late luteal phase (Figure 4
), but the increase was significantly larger in cells obtained from the late luteal phase (P = 0.017).
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Discussion
Detailed studies on the size distribution of small and large luteal cells have shown that in some primate species, such as the rhesus monkey, distinct cell populations can be delineated by size (Brannian and Stouffer, 1991b
). In other primate species, for example the marmoset monkey, a continous distribution of size exists (Brannian and Stouffer, 1991a). In the present study two distinct bands of steroid producing cells were isolated from human CL of both early and late luteal phase. Since these cells differed in both density and cell size, the two bands most likely represent enriched fractions of large and small luteal cells. Similar size and density relation of human luteal cells have been reported to exist in CL tissue from mid luteal phase (Retamales et al., 1994).
A change of relative proportions of small and large luteal cells throughout the life-span of the CL has been suggested to exist in the bovine and human CL (Lei et al., 1991). It is not clear whether this change is due to hypertrophy of the small cells or differentiation of small cells into large cells. In the sheep, experimental data have suggested that luteinizing hormone (LH)/HCG stimulation results in conversion of small luteal cells to large (Farin et al., 1988). With the use of an image analysis system in a study of the human CL the ratio of small to large luteal cells in tissue sections was found to progressively decline from 9:1 in early phase to 3:1 in CL of late phase (Lei et al., 1991). In contrast, a lower and almost stable ratio was found by the method of visual counting of randomly chosen cells (Retamales et al., 1994). In the present study the proportional yield of SCLC increased with the age of the CL. The SCLC/LCLC cell ratio in our cell fractions increased from 1.3 in CL of early luteal phase to 4.2 in the late and this was due mostly to decreased numbers of LCLC during late luteal phase. The decline of the number of LCLC is consistent with findings in the CL of the rhesus monkey (Brannian and Stouffer, 1991a) but differ with one study of human CL tissue (Lei et al., 1991). We can only speculate about the reason for the disparities in the ratio shift of human luteal cells. In the present study we have used cells after separation by enzymatic digestion and cell density sedimentation and not actually counted cell distribution in the tissue. It is possible that the extraction of specific cell types vary during the course of the luteal phase, due to the differences in tissue organization, vascularization, and fibrous content in young and old CL, which would result in the retention of one particular cell type at any stage. It is also important to bear in mind that in a study where cells are counted in tissue blocks (Retamales et al., 1994) the cell distribution may differ depending on the specific area of the CL being analysed. In the human CL, relatively more small luteal cells are situated in the periphery of the corpus luteum, which is mainly made up of the infolded theca–lutein area. In the present study we have studied enriched fractions of steroid-producing cells with >75% steroidogenic cells obtained from the whole CL. It is reasonable to assume that the SCLC fraction of cells has a larger contamination of endothelial cells, as well as immune cells, since their densities are similar. This latter fact can partly account for the rising number of cells in the fractions from the late CL, with the reported simultaneous influx of immune cells (Brännström et al., 1994; Brännström and Fridén, 1997; Duncan et al., 1998). In a recent study, the numbers of white blood cells contaminating human steroidogenic luteal cells obtained by procedures similar to ours was 20% (Castro et al., 1998). The presence of a varying portion of leukocytes and/or endothelial cells may present a explanation for the variability we observed in the progesterone synthesis of SCLC, considering that both cell types secrete factors that influence steroidogenesis in the human CL in vitro (Emi et al., 1991; Apa et al., 1998; Castro et al., 1998; Hashii et al., 1998).
In short-term cultures after 48 h, we noted a three-fold higher protein content for SCLC compared with LCLC (Table I
). It is possible that protein debris attached to viable cells are enriched to a larger degree in this high-density fraction. In prolonged culture, there was a continuous increase in protein content in the cells over 9 days. This increase may be due to proliferation of contaminating fibroblasts and/or endothelial cells, but may also partly represent an increase in total intracellular protein during culture conditions. This has been reported earlier in large luteal cells of the rat during in-vivo luteal development (McLean et al., 1992). Estimation of DNA or thymidine incorporation would in this context be of benefit in discriminating between cellular development and proliferation.
Under our conditions LCLC showed a higher basal progesterone secretion than did SCLC. The progesterone synthesis was about three-fold higher in the LCLC, which is comparable with earlier findings of about five-fold higher concentrations in ovine large luteal cells (Rodgers and O'Shea, 1982; Rodgers et al., 1983) and in accordance to previous studies on large and small luteal cells from the human ovary (Ohara et al., 1987; Retamales et al., 1994). The cellular mechanisms responsible for this difference in steroidogenic capacity in vivo and during in-vitro conditions with added serum may be a higher content of cholesterol side-chain cleavage enzyme, sterol carrier protein-2 (McLean et al., 1992), expression of StAR (Chung et al., 1998), or a greater binding of low-density lipoproteins (LDL) (Brannian et al., 1995). Contrary to large cells, both ovine and human small luteal cells secrete limited basal amounts of progesterone, but have the ability to respond to LH/HCG stimulation with increased progesterone production (Fitz et al., 1982; Ohara et al., 1987; Retamales et al., 1994). It is certainly possible that proteolytic digestion, even using collagenase, may impair the function of hormone receptors, i.e. the LH receptor, on the cell surface. It is difficult to speculate about a differential effect of the enzymes on the same hormone receptor on different luteal cell types. Differences in responsiveness are more likely to be explained by differences in receptor numbers. Such a difference has been observed in a number of species (Gebarowska et al., 1997; Takao et al., 1997; Mamluk et al., 1998). However, in our experimental model, both cell types exhibited a dose-dependent increase in progesterone secretion in response to HCG, presumably via the LH receptor. Interestingly, LCLC from late luteal phase demonstrated a greater response to HCG, compared with the cell fractions from the early luteal phase. This finding is in accordance with results obtained in the macaque monkey (Brannian and Stouffer, 1991b). In SCLC this difference in response to HCG in relation to luteal age was not present. The stimulatory effect of PGE2 did not differ according to cell size or luteal phase. It could be postulated that during the life-span of the CL certain steroid-producing cells become prepared for CL rescue as demonstrated by their increased susceptibility to respond to HCG and not to PGE2.
One objective of the study was to shed light on the process of CL rescue and regression. Long-term culture of purified luteal cells would indicate whether rescue and regression are dependent on factors external or intrinsic to the cells. A major finding of this study is that luteal cells from the late luteal phase increase their basal progesterone formation with time in culture well beyond their assumed time of regression in a non-conception cycle in vivo. In contrast, in luteal cells from the early phase, progesterone formation declined with time in culture. This discrepancy in capability of maintaining steroidogenic function may indicate that during the transition from early to late phase luteal, cells acquire the capability for rescue by HCG in case of pregnancy. Possibly, LH facilitates this transition to longevity by acting during the mid-luteal phase. This could explain why the luteal phase in gonadotrophin-releasing hormone (GnRH) down-regulated cycles, in which LH concentrations are low, is shorter than in clomiphene citrate–human menopausal gonadotrophin (HMG) cycles or natural cycles (Smitz et al., 1988). Other trophic factors in the human CL, such as integrin and fibronectin, also display phase-dependent changes (Honda et al., 1997) and may represent an explanatory model for this intrinsic change in the CL cells rending them longevity. Our data are thus compatible with the view that human luteolysis is an active process involving cellular and/or biochemical elements.
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Acknowledgments |
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We thank Professor Gregory Erickson, University of San Diego, for excellent scientific advice in preparation of this manuscript. This study was supported by grants from the Swedish Medical Research Council (grants no 11237 to M.H. and 11607 to M.B.), the Faculty of Medicine at Göteborg University, the Göteborg Medical Association, and the Merchant Hjalmar Svensson Foundation.
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Notes |
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4 To whom correspondence should be addressed at: Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093–0633, USA
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Submitted on December 1, 1998; accepted on May 11, 1999.
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