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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 4 1672-1677
Copyright © 2000 by The Endocrine Society

Original Studies

Changes in Insulin-like Growth Factor-Binding Protein-3 Messenger Ribonucleic Acid in Endothelial Cells of the Human Corpus Luteum: A Possible Role in Luteal Development and Rescue

Hamish M. Fraser, Stephen F. Lunn, Holly Kim, W. Colin Duncan, Faye E. Rodger, Peter J. Illingworth1 and Gregory F. Erickson

Medical Research Council Reproductive Biology Unit (H.M.F., S.F.L., W.C.D., F.E.R., P.J.I.), Center for Reproductive Biology, Edinburgh EH3 9ET, United Kingdom; and Department of Reproductive Medicine (H.K., G.F.E.), University of California at San Diego, La Jolla, California 92093-0674

Address correspondence and requests for reprints to: Dr. H. M. Fraser, Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, United Kingdom. E-mail: h.fraser{at}ed-rbu.mrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
In the human menstrual cycle, extensive angiogenesis accompanies luteinization; and the process is physiologically important for corpus luteum (CL) function. During luteolysis, the vasculature collapses, and the endothelial cells die. In a conceptual cycle, the CL persists both functionally and structurally beyond the luteoplacental shift. Although luteal rescue is not associated with increased angiogenesis, endothelial survival is extended. Despite the central role of the luteal vasculature in fertility, the mechanisms regulating its development and demise are poorly understood. There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function. Here, we have found that IGFBP-3 messenger RNA is expressed in the endothelium of the human CL and that the levels of message change during luteal development and rescue by human CG. The signal was strong during the early luteal phase, but it showed significant reduction during the mid- and late luteal phases. Interestingly, administration of human CG caused a marked increase in the levels of IGFBP-3 messenger RNA in luteal endothelial cells that was comparable to that observed during the early luteal phase. We conclude that endothelial cell IGFBP-3 expression is a physiological property of the CL of menstruation and pregnancy. These observations raise the intriguing possibility that the regulated expression of endothelial IGFBP-3 may play a role in controlling angiogenesis and cell responses in the human CL by autocrine/paracrine mechanisms.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN THE HUMAN (1), as in ruminants (2, 3) and nonhuman primates (4), the vascular endothelial cells of the ovulated follicle undergo an intense period of proliferation during its transformation to a fully functional corpus luteum (CL). This process of angiogenesis continues throughout the luteal phase, albeit at a lower rate. Whereas in most tissues, the life span of endothelial cells is 1–2 yr, in the CL of the nonfertile cycle, most endothelial cells die in the space of a few weeks during the process of luteolysis. After conception, embryo-derived human CG (hCG) rescues the CL from luteolysis, and the CL of pregnancy then persists for up to 6 months (5), with maintenance of the luteal endothelium. Luteal rescue does not involve recognizable changes in angiogenesis in the human (1), macaque (4), or nonprimate CL (6). Because luteal endothelial cells must survive for relatively long periods of time during pregnancy, it is clear that the mechanism underlying angiogenesis in the CL is a highly regulated process. Because the CL has a crucial role in establishing and maintaining early pregnancy, understanding the physiological mechanisms that regulate luteal endothelial cell proliferation in the CL is a major objective in reproductive research. Despite its importance, however, little is known about the regulation of the vasculature of the CL in any species.

There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function (7, 8, 9). Previous findings suggest that IGFBP-3 may be one such factor of physiological importance in the vascular system of the CL. In the rat ovary, IGFBP-3 is localized predominantly to the vascular endothelial cells but only in regressing CL (10). This suggested that the induction of IGFBP-3 gene activity in the endothelium might be involved in luteolysis in this species. Further, in support of its role as an intrinsic luteal regulator, IGFBP-3 gene expression has also been shown to be induced in the endothelial cells of the marmoset CL; however, in contrast to the rat, IGFBP-3 was expressed throughout the luteal phase and declined during luteolysis (11). Collectively, these observations, demonstrating the tissue-specific expression of IGFBP-3 in the endothelium of the CL, support the hypothesis that differential regulation of IGFBP-3 expression might mediate important luteolytic and luteotrophic changes in the vascular system of the rat and primate, respectively.

IGFBP-3 messenger RNA (mRNA) has been shown to be localized to endothelial cells in the human ovary, but temporal changes in its expression were not determined because only four corpus lutea (CL) were examined (7). To begin to address the clinical relevance of IGFBP-3 in the CL, we investigated the pattern of expression of IGFBP-3 mRNA in human corpora lutea during luteinization, luteolysis, and rescue by hCG.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Treatments and collection of tissue

Corpora lutea were enucleated at the time of hysterectomy, as described previously (1, 12, 13). All women were healthy, 32–45 yr old, with regular menstrual cycles, and had not received any form of hormonal treatment for at least 3 months before taking part in the study. The date of the preovulatory luteinizing hormone (LH) surge was determined by measuring LH concentrations in serial early-morning urine samples collected before operation (13). On this basis, three CL were classified as early luteal (LH+2 to LH+4 days), five as midluteal (LH+6 to LH+9 days), and four as late luteal (LH+11 to LH+19 days). In addition, five women were given im injections of hCG (Profasi, Serono Laboratories, Inc., Welwyn Garden City, UK) from LH+7, in daily doubling doses, starting at 125 IU and continuing for 6–8 days until surgery (13). This regimen has been shown to rescue the CL and reproduce the hormonal changes of early pregnancy (14). Corpora lutea were enucleated from the ovary by blunt dissection, the tissue immediately divided into radial blocks, and a portion fixed in 4% paraformaldehyde for 24 h. These corpora lutea were further used in studies to determine changes in other factors associated with control of luteal cell function described elsewhere (1, 12, 13).

An endometrial biopsy was also obtained to assist luteal staging by tissue morphometry. In all cases, morphological dating of the luteal-phase endometrium (15) was used to confirm the luteal-phase classification. Plasma was taken before surgery, and progesterone concentrations were measured using a standard RIA (13). The study was approved by the Reproductive Medicine Branch of the South-East of Scotland Medical Ethics Committee, and informed consent was obtained from all patients before tissue collection.

In situ hybridization

In situ hybridization was performed using a complementary RNA probe for human IGFBP-3, as described previously (11). Paraffin sections (5 µm) were mounted onto poly-L-lysine-coated glass slides, secured in racks, and transported in boxes to San Diego for mRNA localization by in situ hybridization. Sections were deparaffinized in xylene and hydrated through descending concentrations of ethyl alcohol. Sections were digested in 0.32% proteinase K (Roche Molecular Biochemicals, Indianapolis, IN) for 20 min at 37 C, acetylated, and dehydrated. Sense and antisense probes were prepared using an RNA transcription kit (Stratagene, La Jolla, CA) and labeled with 35S uridine 5'-triphosphate (Amersham Pharmacia Biotech, Arlington Heights, IL). The synthesized probes were purified from free bases by using Quick Spin columns (Roche Molecular Biochemicals). Hybridization was performed with the probes at 107 cpm/mL hybridization solution containing 50% (vol/vol) formamide, 0.3 mol/L NaCl, 10 mmol/L Tris, 1 mmol/L EDTA, 0.05% yeast transfer RNA, 10 mmol/L dithiothreitol, 1 x Denhardt’s solution, and 10% Dextran sulphate. Two sections per slide on two slides were exposed to the antisense probe, and two sections on a single slide were exposed to the sense sequence. Each slide was hybridized with 80 µL hybridization solution on a 60-C slide warmer overnight. Slides were rinsed in SSC and then treated with ribonuclease A (20 µg/mL) for 30 min at 37 C to remove all excess probe, desalted in descending concentrations of SSC, washed in 0.1 x SSC at 75 C, and dehydrated. Dry slides were exposed to Hyperfilm-Max (Amersham Pharmacia Biotech) for 4 days, then dipped in NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY), and exposed for 24 days, at 4 C, in light-tight slide boxes. Slides were developed with D-19 developer (Eastman Kodak Co.) for 3.5 min at 4 C and subsequently fixed. The sense and one antisense slide were counterstained with hematoxylin, whereas the remaining antisense slide was stained with hematoxylin and eosin (Richard-Allan, Richland, MI).

Analysis

The slides were analyzed under both light- and darkfield conditions, assessment being made of the degree and frequency of expression in endothelial cells according to blood vessel size. Vessels with one to three visible endothelial cells were considered capillaries, whereas those in which a lumen was visible, often containing erythrocytes and surrounded by more than four endothelial cells, were classed as microvessels (and included venules and arterioles). The intensity of the hybridization signal was scored as follows: -, no expression above tissue background; +, low expression; ++, moderate expression; +++, high expression. Two further observers scored the sections ‘blind’. Comparison of the results between the three observers showed excellent agreement. Differences between groups, with respect to level of mRNA expression, were analyzed using the Mann-Whitney U test, significance being ascribed at a level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Plasma progesterone concentrations

The classification of corpora lutea by serial urinary LH measurement agreed with the luteal phase dating of endometrial biopsies. The plasma progesterone concentrations were 22 ± 12 (mean ± SEM) nmol/L in the early luteal samples, 41.0 ± 9.9 nmol/L in the midluteal samples, and 19.2 ± 12.9 nmol/L in the late luteal samples. After luteal rescue by exogenous hCG, plasma progesterone concentrations increased to 52.6 ± 1.5 nmol/L.

Expression of IGFBP-3 mRNA

Figure 1Go is of a low magnification and depicts the distribution of IGFBP-3 mRNA in a typical early human CL with adjacent stromal tissue. The IGFBP-3 mRNA is found predominantly in the vascular system, being strongly expressed in the peripheral stromal microvessels and the straight capillaries of the CL proper. Heterogeneity in labeling is evident, both in the CL and stroma, with some blood vessels appearing negative for IGFBP-3. Higher magnification of another early-luteal-phase CL (Fig. 2Go) shows that the expression is occurring in the endothelial cells lining the microvessels and capillaries. Once again, heterogeneity is evident. In this example, the distribution of labeling suggests radiation of the capillaries from the microvessel from which they have originated. IGFBP-3 mRNA was not detected in the other cell types within the CL (Fig. 2Go). Absence of expression in the granulosa and theca lutein cells is also evident in the midluteal section shown in Fig. 3Go. This specimen illustrates how expression is high in some vessels at this stage, whereas in other luteal microvessels, expression is virtually absent.



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  		Figure 1. Low-power light- (a) and darkfield (b) photomicrographs of a portion of the CL, together with adjacent stromal tissue (S). Expression of IGFBP-3 mRNA is occurring in most, but not all, of the microvessels (arrows) and capillaries of both the CL and stromal tissue. The scale bar represents 200 µm.

 


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  		Figure 2. Light- (a) and darkfield (b) photomicrographs showing expression of IGFBP-3 mRNA in the human CL from the early luteal phase. Note the microvessel (large arrow) lying across the field of view, with multiple smaller capillaries (arrowheads) emanating in a spoke-like formation. GL, Granulosa lutein cells. The scale bar represents 100 µm.

 


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  		Figure 3. Lightfield photomicrograph showing expression of IGFBP-3 mRNA in human CL during the midluteal phase, showing a microvessel lying in the centre with smaller microvessels (arrows) running above it. TL, Theca lutein cells. Note the strong expression in the central vessel and virtual absence of expression in the remaining vessels. The scale bar represents 50 µm.

 
IGFBP-3 mRNA in the corpora lutea collected during the nonconceptual cycle showed clear variations depending on the stage of the luteal phase at which it was collected (Fig. 4Go). It was highly expressed during the early luteal phase (Figs. 4Go and 5aGo); being evident in most of the endothelial cells throughout the CL, e.g. at the periphery, throughout the theca/granulosa lutein layer, and in the luteal cavity (Fig. 5aGo). IGFBP-3 mRNA was expressed at a significantly lower level in corpora lutea during the mid- (P < 0.025) and late luteal phase (P < 0.04) (Fig. 4Go); the hybridization signal was low or undetectable in the vast majority of endothelial cells located in the capillaries in the granulosa lutein layer, although a strong signal was seen in some endothelial cells in the microvessels juxtaposed to the theca lutein cells (Fig. 5bGo). The CL during the late luteal or regressing stage showed low levels of expression (Fig. 4Go), and the pattern of expression seemed similar to that seen in the midluteal phase (Fig. 5cGo). There was a significant increase (P < 0.04) in the level of expression of IGFBP-3 mRNA in corpora lutea during pharmacologically-induced luteal rescue, compared with the late-luteal-phase corpora lutea (Fig. 4Go). In these so-called rescued corpora lutea, the regressive changes in the vascular system seen in late-luteal-phase corpora lutea seemed to have been reversed, in that the vasculature was prominent, with many luminal vessels coursing through the CL. The expression of IGFBP-3 mRNA was very high in most endothelial cells of the entire rescued CL (Fig. 5dGo), essentially similar to the level and pattern of expression observed in corpora lutea at the early luteal phase (compare Fig. 5Go, a and d).



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  		Figure 4. Intensity of expression of IGFBP-3 mRNA in the individual specimens of the early, mid-, late, and rescued CL.

 


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  		Figure 5. Typical patterns of expression of IGFBP-3 mRNA in the early, mid-, late, and rescued CL. The scale bar represents 200 µm.

 
Where present, changes in IGFBP-3 mRNA expression in stromal vessels generally seemed to reflect the stage of the luteal phase. No specific hybridization above background was detected using the IGFBP-3 complementary RNA sense probe.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have established, for the first time, that the gene encoding IGFBP-3 is expressed in a cyclical manner in the endothelial cells of the human CL during luteal development and hCG-induced rescue. In the vascular system, we have found that the highest levels of IGFBP-3 expression occur when the endothelial cells are undergoing extremely rapid proliferation during the process of angiogenesis in the early luteal phase of the menstrual cycle. We found also that the lowest levels of IGFBP-3 mRNA occur during the mid- to late luteal phases of the cycle, when endothelial cell proliferation is declining and the blood capillaries within the CL begin to show reduction in cross-sectional area (16). One of the most interesting observations is the recurrence of expression of IGFBP-3 mRNA at high levels within these endothelial cells after hCG-treatment. This is an event associated with the continued development and function of the vascular system. Collectively, these findings lead us to propose that intrinsic endothelial cell IGFBP-3 may have an autocrine role in regulating the growth and development of the vascular system, because it shows differential expression during luteolysis and luteal rescue.

The high level of expression in the early luteal phase suggests a positive role for IGFBP-3 during the formation of the CL, when endothelial cell replication is at its highest (1). This agrees with our previous report in the marmoset (11) and suggests IGFBP-3 may regulate angiogenesis and luteal function rather than endothelial cell death and luteolysis, as is apparently the case in the rat (10). The lower expression during the mid- and late luteal phases may be indicative of an attenuation of this role, as endothelial cell proliferation is declining. That a decline in IGFBP-3 expression is associated with naturally occurring luteolysis is supported by the observations in the CL of the marmoset, in which the IGFBP-3 mRNA was either absent or confined to the microvessels of the CL during the late luteal phase (11). These changes are the result of differences in IGFBP-3 expression between endothelial cells and not a reflection of reduction in numbers of endothelial cells; we have shown previously that numbers of endothelial cells in the early luteal phase were significantly lower than in the mid- and late luteal phases by studying the majority of corpora lutea used in the present study (1).

It was of particular interest that IGFBP-3 expression was elevated in corpora lutea from women who had been treated with hCG from the midluteal phase to mimic the rescue of the CL during early pregnancy. In the hCG-treated CL, endothelial cell proliferation shows no increase (1, 4), and endothelial cell numbers are similar to those in the mid- and late luteal phases (1); however, the function of both the hormone-producing cells and endothelial cells is maintained during this period, at a time when they would normally undergo apoptosis in the absence of pregnancy (17, 18, 19). It is tempting to propose that IGFBP-3 is involved as a component of a survival mechanism for the endothelial cells at this time.

Defining the precise role of IGFBP-3 in luteal endothelial cells is likely to be difficult, because it is clear from studies in other systems that IGF/IGFBP interactions can be very complex. IGF acts in a wide variety of organs and tissues to stimulate cell proliferation and amplifies the effects of the tropic hormones on the gonads (20, 21). In most situations, the action of IGFBP-3 is inhibitory, because, when bound to IGFBP-3, IGF is unable to interact with its receptor. Indeed, most of the information described previously for IGF/IGFBP-3 interactions in the ovary have indicated an inhibitory function, e.g. IGFBP-3 has been shown to inhibit the progesterone response to IGF in human granulosa cells (22). However, depending on the tissue site and on the experimental paradigm, IGFBP-3 has been reported to both facilitate or inhibit IGF action in different cells (20). Previous studies on the ovary have focused on the paracrine function of IGFBP-3, and it is possible that an autocrine role within the luteal endothelial cells may be quite different. An apparently divergent role for the same molecule in controlling angiogenesis and blood vessel stability is not without precedent. Within the angiopoietin family, for example, angiopoietin-2 has a positive role in angiogenesis in the presence of vascular endothelial growth factor; but when vascular endothelial growth factor is absent or reduced, angiopoietin-2 acts to destabilize endothelial cells (23, 24). It may also be of relevance that, in some tissues, endothelial cell IGFBP-3 has been implicated in the transport of IGFs from the bloodstream to the subendothelial cells (25), suggesting a positive role in tissue metabolism. IGF-1 receptors have been identified in the human CL (8, 26), although their precise location has yet to be established. Because IGF-1 may function to increase progesterone production, such a positive role of IGFBP-3 might serve to contribute to the increased progesterone production during early pregnancy.

Although IGFBP-3 mRNA expression was observed in the endothelial cells in the majority of microvessels and capillaries, the reason for the absence of expression in others is unclear. No morphological explanation for the failure of some cells to show hybridization was apparent. Although the reasons for the heterogeneity in expression remain to be established and will require further investigation, they may relate to differential maturity of individual endothelial cells. It is also known that endothelial cells throughout the vasculature do not constitute a homogeneous population of cells (27), and differences in function may occur within, as well as between, vascular beds. Additionally, further studies are required to determine whether expression of the IGFBP-3 mRNA is continuously associated with its translation into a functional protein or whether there are translational restrictions.

In conclusion, our data are consistent with the hypothesis that IGFBP-3 may play an autocrine/paracrine role in CL regulation in the primate ovary. The identification of the IGFBP-3 mRNA in the endothelial cells suggests a specific role of the protein product in the microvasculature of the CL. Further studies will be required to address the mechanisms controlling the cyclical regulation of IGFBP-3 expression within the human CL and to address its specific role in the development and demise of this important endocrine tissue during the menstrual cycle and pregnancy. Because maintenance of luteal function is essential for continuation of pregnancy, and inhibition of angiogenesis has been shown to have an antifertility effect in the rodent (28) and nonhuman primate (29), the possibility that IGFBP-3 may have a role in regulation of the endothelial cells should add impetus to such studies, with the further potential for development of agents that stimulate or inhibit angiogenesis in the CL.


   Acknowledgments
 
We thank Mike Millar and Pawlina Largue for expert technical assistance.


   Footnotes
 
1 Present address: Department of Obstetrics and Gynaecology, University of Sydney, Westmead Hospital, Sydney, 2145, Australia. Back

Received July 22, 1999.

Revised December 3, 1999.

Accepted December 15, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals