1 MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK, and 2 Department of Obstetrics and Gynaecology, Westmead Hospital, University of Sydney, NSW, Australia
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Abstract |
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Key words: angiogenesis/BrdU/corpus luteum/endothelial cells/Ki67
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Introduction |
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The high rates of endothelial cell proliferation which occur during formation of the corpus luteum have been well characterized in ruminants (Jablonka-Shariff et al., 1993; Zheng et al., 1994
), macaques (Christenson and Stouffer, 1996
) and women (Rodger et al., 1997
). These studies also demonstrated that proliferation decreases as the luteal phase proceeds, and remains low during functional luteal regression. In ruminant corpora lutea the network of fine capillaries required for optimal production of steroid hormones during the luteal phase (Redmer and Reynolds, 1996
) is replaced by larger microvessels (Fraser et al., 1998
) during luteal regression (Modlich et al., 1996
). However, luteolysis is regulated by different mechanisms in primates and ruminants, and there are no data regarding cell proliferation or vascular status during structural luteal regression in primates.
Induction of luteolysis with pharmacological agents has a number of advantages over the collection of spontaneously regressing luteal tissue; tissue can be collected at known time points after induction of luteolysis, and different luteolytic agents can be used in order to gain insight into the mechanisms controlling luteal involution. Luteal regression can be induced in marmosets in two ways; by administration of a gonadotrophin-releasing hormone (GnRH) antagonist, which deprives the corpus luteum of luteinizing hormone (LH) support (Hodges et al., 1988), or by administration of a prostaglandin (PG) F2
analogue, which is thought to act directly at the level of the corpus luteum (Summers et al., 1985
)
This study examined cell proliferation and vascular morphology in a commonly used non-human primate model, the marmoset monkey, and focused on cell proliferation and vascular changes during spontaneous and induced luteal regression. Specific aims were to: (i) identify cell proliferation using Ki67 and bromodeoxyuridine (BrdU); (ii) investigate changes in vascular morphology by determining the temporal and morphological distribution of endothelial cells using von Willebrand factor VIII antigen (vW) as a marker; and (iii) identify proliferating cells by co-labelling with BrdU and the steroidogenic cell marker 3ß-hydroxysteroid dehydrogenase (3ßHSD), or the endothelial cell marker vW.
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Materials and methods |
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Additional animals were administered i.v. BrdU (Boehringer Mannheim, Lewes, Sussex, UK) by slow infusion at a dose of 20 mg/ml saline and ovaries were collected 1 h later. Ovaries containing corpora lutea were collected on luteal days 24 (n = 3), during functional regression on luteal day 18 (n = 3) and during structural regression on luteal days 2225 (n = 3).
Marmosets were also treated on day 9 of the luteal phase with either a GnRH antagonist, Antarelix® (N-Ac-D-Nal1,D-pCl-Phe2, D-Pal3,D-(hic)6,-Lys(iPr)8,D-Ala10; Deghenghi et al., 1993) 1 mg/kg s.c (Young et al., 1997), or a PGF2
analogue cloprostenol (Planate®, Coopers Animal Health Ltd, Crewe, Cheshire, UK) 1 µg i.m.
Ovaries containing corpora lutea were collected 12 h (n = 3 per treatment) or 24 h (n = 4 per treatment) after administration of luteolytic agents. PGF2 does not induce luteal regression during the early luteal phase, and is only luteolytic after luteal days 7/8. GnRH antagonist is anti-steroidogenic during the early luteal phase and was therefore used as a comparison for induction of luteolysis. The mid-luteal phase was chosen to avoid superimposing induced luteolysis on spontaneous, naturally occurring luteal regression. Corpora lutea collected 48 h after administration of PGF2
on luteal days 8 and 9, looked very similar to corpora lutea collected 24 h after treatment with PGF2
.
Ovaries were fixed in 4% (w/v) buffered paraformaldehyde (BDH Laboratories Ltd, Poole, Dorset, UK) for 24 h prior to embedding in paraffin according to standard procedures. Sections (4 µm) were mounted on poly-L-lysine coated (Sigma, Poole, Dorset, UK) slides, air-dried at 56°C and subjected to immunohistochemistry.
Immunohistochemistry
Endothelial cells were identified as previously described (Rodger et al., 1997) but with the following modifications. Primary antibody (von Willebrand factor (vW), polyclonal rabbit anti-human immunoglobulin G (IgG) (Dako, High Wycombe, Bucks, UK) was diluted 1:250 in Tris-buffered saline (TBS, 0.05 mol/l Tris, NaCl 9 g/l) and negative control sections were treated with normal rabbit serum (NRS) (SAPU, Carluke, UK) also diluted 1:250 in TBS before being incubated for 18 h at 4°C. Visualization was with avidinbiotin complex conjugated to alkaline phosphatase (ABCAP, Vector Laboratories, Peterborough, UK) and Vector Red (Vector Laboratories) according to the manufacturer's instructions.
Analysis of proliferation depends on assessment of the number of cells undergoing mitosis in a given population, but the likelihood of observing mitotic spreads in histological sections is very low (O'Shea et al., 1986). More sensitive techniques for evaluating proliferation exploit characteristics of the cell cycle. Ki67 antigen is a nuclear, non-histone protein routinely used to identify proliferating cells (Gerdes et al., 1984
; Bruno and Darzynkiewicz, 1992
). Ki67-positive cells were identified as described previously (Rodger et al., 1997
). Primary antibody (Ki67 antigen, monoclonal mouse anti-recombinant protein, Novocastra, Peterborough, UK) was diluted 1:50 in TBS and negative control sections were treated with mouse IgG (Vector Laboratories) also diluted 1:50 in TBS before incubation for 18 h at 4°C. Detection of the primary antibody was carried out in the same manner as for vW.
DNA synthesis occurs before mitosis, and BrdU can be incorporated into new DNA in place of thymidine and visualized using standard immunocytochemical techniques (Boulton and Hodgson, 1995). BrdU incorporation in vivo was used to confirm and extend data about proliferation rates obtained by Ki67 immunohistochemistry. Sections were deparaffinized in xylene and hydrated in 100, 95 and 70% ethanol. Endogenous peroxidase activity was blocked by incubating in 3% (v/v) hydrogen peroxide in methanol (BDH Laboratories Ltd) for 20 min at room temperature. BrdU antigen retrieval was by exposure to two 5 min cycles of microwave irradiation at 700 W in glycineHCl buffer pH 7.4 (0.2 mol/l glycine, 0.2 mol/l HCl). Buffer was replenished to the original volume after each 5 min cycle of microwaving. Primary antibody (mouse monoclonal antibody clone BMC 9318 to BrdU, Boehringer Mannheim) was diluted 1:30 in TBS. Negative control sections were treated with mouse immunoglobulin G (Vector Laboratories) which was also diluted 1:30 in TBS before all sections were incubated for 18 h at 4°C. After washing with TBS, rabbit anti-mouse IgG (Dako) was applied at a dilution of 1:60 in blocking medium (20% NRS, 5% bovine serum albumin w/v in TBS) for 30 min at room temperature. This was followed by three washes with TBS and incubation with mouse peroxidase anti-peroxidase (mouse PAP; Dako) diluted 1:100 in TBS for 30 min at room temperature. Visualization was by application of 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical Co Ltd, 0.04% w/v and 0.01% hydrogen peroxide in 0.05 mol/l Tris).
Proliferating endothelial cells were identified by vW and BrdU dual immunostaining. Von Willebrand immunocytochemistry was as described above, sections were then washed in water before proceeding with BrdU detection as described above.
The nature of BrdU-positive non-endothelial cells was investigated by dual immunostaining with 3ßHSD primary antibody (polyclonal rabbit anti-human 3ßHSD, gift of Prof Van Luu-The, the CHUL Research Centre, Quebec, Canada) in order to determine whether these cells were steroidogenic. BrdU detection proceeded as described above. After visualization, sections were washed with water before application of the 3ßHSD primary antibody which was diluted 1:300 in TBS. Negative control sections were treated with NRS (SAPU) also diluted 1:300 in TBS. Primary antibody and negative control sections were then incubated for 18 h at 4°C. Visualization was with ABC-AP and Vector Red (Vector Laboratories) made up according to the manufacturer's instructions. Visualization substrates can sometimes mask the colour of a second dye used in co-localization studies. Vector Red is fluorescent and not masked by DAB.
Analysis of data
Area of vW immunostaining was measured using NIH Image 1.57 software which converted the colour image to black and white. The optical density value that corresponded to the lightest vW immunostaining was determined for each section, then all areas above that threshold were measured. Negative control sections were measured in the same way, and areas with optical density values equal to or higher than the threshold for vW immunoreactivity were subtracted from the mean value for the corresponding experimental section. Four fields of view of luteal tissue were randomly selected and measured for each ovarian section, then their mean taken as being representative for that animal. Measuring more than four unit areas, from different ovarian sections, did not significantly alter the mean. The area of vW immunoreactivity (µm2) was calculated for each animal and expressed as mean area ± SEM for each experimental group.
Ki67 immunocytochemistry was quantified using a highly optimized microscope environment (Brugal et al., 1992) which was used previously and allowed comparison with data from an earlier study (Young et al., 1997
). Four fields of view of luteal tissue were randomly selected and the mean of the four unit areas was taken as being representative for that animal. Measuring more than four unit areas from serial sections did not alter the calculated mean. Cells were classified as being either steroidogenic or non-steroidogenic, and also Ki67-positive or negative. Cells were classified as being steroidogenic or non-steroidogenic by morphological, not functional, criteria. Steroidogenic cells were >12 µm in diameter with regular circular outlines, abundant cytoplasm and circular nuclei. Cells with these morphological characteristics were also 3ßHSD-positive in this study. Non-steroidogenic cells were elongated with a long axis of 48 µm, large nuclei and little cytoplasm. Cells with this morphology were also vW-positive in this study, so the majority of these were endothelial cells, and a minority were other cell types; fibroblasts, pericytes and immigrant leukocytes (Lei et al., 1991
).
The numbers of Ki67-positive steroidogenic and non-steroidogenic nuclei per unit area were expressed as a mean ± SEM for each experimental group. The mean percentages ± SEM of Ki67 positive steroidogenic and non-steroidogenic cells were also calculated for each experimental group.
BrdU immunohistochemistry was quantified by scoring every cell in the field of view of a x40 objective lens as being either steroidogenic or non-steroidogenic (using the morphological criteria described above) and either BrdU-positive or -negative. Four fields of view were scored for each corpus luteum, and the mean scores of the four unit areas were taken as being representative for that animal. Data were expressed as mean percentages ± SEM of BrdU-positive steroidogenic and non-steroidogenic cells for each experimental group.
BrdU and vW co-labelling was quantified by scoring every BrdU-positive cell in the field of view of a x40 lens, and classifying it as being either vW-positive or -negative. Four fields of view were scored for each corpus luteum, and the mean scores of four unit areas were taken as being representative for that animal. The mean ± SEM was then determined for each experimental group. BrdU and 3ßHSD quantification was obtained in the same way.
Progesterone assay
Progesterone concentrations were determined by radioimmunoassay as described previously (Smith et al., 1990). The means of each group were expressed ± SEM.
Statistical analysis
Data from untreated mid-luteal phase corpora lutea were compared with corpora lutea after induced regression with either PGF2 or GnRH antagonist in a one-way analysis of variance using treatment as a between subject variable. Differences in numbers of immunopositive cells in early, mid-, late and follicular phase corpora lutea were also investigated by one-way analysis of variance using stage of the luteal phase as a between subject variable. Confidence intervals for the differences between means were evaluated by using Fisher's least significant difference (LSD) procedure. P < 0.05 was considered to be significant.
Percentages of proliferating cells in corpora lutea from the early, mid-, late and follicular phases were also examined by one-way analysis of variance using stage of the luteal phase as a between subject variable. Percentage data were log-transformed before conducting one-way analysis of variance. Differences were evaluated by using Fisher's LSD procedure; P < 0.05 was considered to be significant. Mean differences in BrdU and vW co-localization were determined by paired t-tests, and P < 0.01 was considered to be significant.
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Results |
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The percentage of steroidogenic cells which were Ki67 immunopositive did not change significantly through the luteal phase nor during spontaneous luteal regression (Figure 10). The percentage of Ki67-positive non-steroidogenic cells decreased significantly from 8.4 ± 0.8% during the early luteal phase, to 0.6 ± 0.3% (P < 0.01) during functional luteal regression and 2.2 ± 0.5% (n = 3, P < 0.01) during structural luteal regression.
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In the early luteal phase, 64.6% of BrdU-positive cells were also vW immunopositive (17 ± 3.5, mean ± SEM, Figure 9). This decreased significantly to 0.42 ± 0.2 (P < 0.001) during functional regression, then returned to values similar to those seen in the early luteal phase (15.7 ± 2.5).
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Discussion |
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Immunocytochemistry for vW indicated that during the early and mid-luteal phase vascular morphology was consistent with an extensive network of fine capillaries with a high surface area to volume ratio. During structural luteal regression, however, the area of vW immunostaining remained constant whilst the number of individual blood vessels per unit area decreased. This was based on the assumption that each distinct area of vW immunopositive staining represented one blood vessel, even though it was noted that the same blood vessel may intersect the histological plane of section any number of times, and that some endothelial cells may have been vW immunonegative, thus leading to an underestimation of the amount of vasculature present. Despite these limitations, these data support the hypothesis that there is an initial period of intense angiogenesis during formation of the corpus luteum, then a period of vascular regression during functional luteolysis. Endothelial cell proliferation increases during structural regression, suggesting a second period of angiogenesis in order to form larger blood vessels suitable for channelling away the cellular debris of structural regression. Macrophages do not proliferate in the human corpus luteum (Gaytán et al., 1998) therefore it is likely that the proliferating non-endothelial, non-steroidogenic cells are fibroblasts. In human corpora albicans, the endothelial area of each vessel increases and the number of blood vessels decreases in comparison with mid-luteal phase values (Suzuki et al., 1998
), indicating a change in human luteal vasculature during structural regression consistent with our data.
The administration of luteolytic agents resulted in functional luteal regression, unchanged proliferation and the maintenance of an extensive capillary network. In contrast, untreated corpora lutea had functional regression accompanied by decreased proliferation, followed by structural regression characterized by a decrease in the number of capillaries and an increase in the number of larger microvessels. These differences may be related to the level of PGF2 receptor expression. Human PGF2
receptor mRNA was lowest during the mid-luteal phase, and increased as the corpus luteum aged (Ottander et al., 1999
). This suggests that PGF2
receptor levels during luteal day 10 in marmosets may have been sufficient to facilitate the inhibition of steroidogenesis, but not high enough to cause regressive structural changes characterized by decreased proliferation and vascular remodelling.
Oxytocin and oxytocin receptor numbers peak in primate corpora lutea during the mid-luteal phase (Khan-Dawood and Dawood, 1999). Oxytocin stimulates the production of PGF2, and also stimulates endothelial cell production of endothelin-1 (ET-1) (Girsch et al., 1996). ET-1 inhibits steroidogenic enzymes and progesterone production in rat granulosa cells (Tedeschi et al., 1992) and inhibits progesterone production by human luteal cells in vitro (Apa et al., 1998
). ET-1 has been demonstrated in porcine (Flores et al., 1995
) and human (Apa et al., 1998
) corpora lutea and values in bovine corpora lutea were highest during regression (Girsh et al., 1996
). It is possible that in primates, oxytocin stimulates PGF2
and ET-1 production during the mid-luteal phase. PGF2
generates the oxygen free radical, hydrogen peroxide (Riley and Behrman, 1991
) which may trigger a cascade of oxygen free radical reactions capable of inhibiting LH-stimulated steroidogenesis in a very short period of time. Mid-luteal phase production of ET-1 might also inhibit progesterone production, and augment the anti-steroidogenic effects of PGF2
. Both PGF2
and ET-1 concentrations increase, but oxytocin production decreases, as the corpus luteum ages, suggesting that although oxytocin might be an instigating factor in functional regression, it is not required to maintain the process.
The number of proliferating non-steroidogenic cells did not change at any time after induction of luteolysis, therefore the significant increase in the area of vW immunoreactive staining after administration of GnRH antagonist can probably be attributed to the dynamics of shrinking tissue. In this scenario the steroidogenic cells die as a result of GnRH antagonist induced LH-depletion (Young et al., 1997), and the remaining blood vessels move closer together, effectively increasing the number of blood vessels in each unit area.
A proportion of cells with the morphological appearance of steroidogenic cells were both Ki67 and BrdU-immunopositive. This was unexpected, since a number of studies have demonstrated that steroidogenic cell numbers do not increase in bovine (O'Shea et al., 1989), ovine (O'Shea et al., 1986
) or human (Lei et al., 1991
) corpora lutea.
This suggests that steroidogenic cells may be in the cell cycle, and thus expressing the cell cycle antigen Ki67, but that they may be arrested in one part of the cell cycle and do not proceed to DNA synthesis or mitosis. Others have described cells with the morphological features of steroidogenic cells positive for proliferation markers in the bovine (Zheng et al., 1994), ovine (Jablonka-Shariff et al., 1993
), macaque (Christenson and Stouffer, 1996
) and human (Rodger et al., 1997
; Gaytán et al., 1998
) corpora lutea, and that these cells did not co-label with the steroidogenic cell marker 3ßHSD. This suggests that cells with the morphological appearance of steroidogenic cells may not produce steroid hormones and express cell cycle antigens simultaneously. Our data support this hypothesis: BrdU-positive cells with the morphological appearance of steroidogenic cells were 3ßHSD-negative. Similarly, the majority of Ki67-positive granulosalutein and thecalutein cells in human corpora lutea did not co-label with 3ßHSD (Rodger et al., 1997
). In the present study, the number of Ki67-positive steroidogenic cells was decreased after induction of luteolysis with either GnRH antagonist or PGF2
, whereas the number of Ki67-positive non-steroidogenic cells remained unchanged. This differential regulation of cell types by luteolytic agents requires further investigation.
High variability within experimental groups may be attributed to the lack of differentiation between the younger (ovulated more recently) and older corpora lutea. In marmosets, ovulation can occur as much as 24 h apart in the same cycle and, therefore, the value derived from one ovary encompassed variablity in luteal age. Our cycle tracking regime was accurate ± 24 h; therefore an ovary collected on day 4 of the luteal phase may in fact have been 3 or 5 days post-ovulation/LH. The Ki67 value derived from each experimental group therefore encompassed variability attributable to cycle tracking. In addition, the large SEM in the early luteal phase group probably reflected the degree of differentiation/luteinization in individual cells, and variable maturation of the corpora lutea. The large SEM in 2025 day old corpora lutea probably reflected variability in the length of the luteal phase. Marmosets had a luteal phase of 20 ± 10 days (mean ± SD, n = 87, range 663 days). The median length of the luteal phase was 18 days, but functional regression could start from day 12 onwards, and progression of functional and structural regression was highly variable. Ovaries collected on luteal day 22 could be beginning structural regression in one animal whilst another animal might have completed structural regression by luteal day 22.
Low numbers of steroidogenic cells were BrdU-positive in the early luteal phase, when granulosa or thecalutein might conceivably be proliferating during the process of transformation from follicle to corpus luteum, but there were no BrdU-labelled steroidogenic cells in the late luteal phase, indicating that steroidogenic cells were not synthesising DNA at this time. The number of BrdU-labelled steroidogenic cells increased during structural luteal regression, but the number of Ki67-positive steroidogenic cells remained constant. One of the first stages of apoptosis can be activation of endogenous endonucleases and fragmentation of DNA, and it is possible that the very early stages of DNA fragmentation might also be accompanied by futile DNA repair processes. Apoptotic glandular prostate cells incorporate BrdU during futile DNA repair when the cells are in the G0 phase (Berges et al., 1993), therefore BrdU-labelled non-proliferating cells might be observed in tissues in which there are also high rates of apoptosis. It is possible that a proportion of the BrdU-labelled steroidogenic and non-steroidogenic cells observed in structurally regressing corpora lutea are not actually proliferating, but are in the initial stages of cell death.
In summary, it is possible to draw three main conclusions: (i) morphological changes in luteal vasculature do not precede functional luteal regression in marmoset corpora lutea. Vascular changes occur after functional regression is completed and progesterone concentrations have fallen to follicular phase values; (ii) the vasculature changes from an extensive network of fine capillaries during the luteal phase to a system of larger blood vessels during luteal regression; and (iii) one component of functional luteal regression is a dramatic reduction in the rate of proliferation.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 1, 1999; accepted on November 19, 1999.