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Endocrinology, doi:10.1210/en.2008-0204
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Endocrinology Vol. 149, No. 10 5024-5034
Copyright © 2008 by The Endocrine Society

Novel Regulated Expression of the SLIT/ROBO Pathway in the Ovary: Possible Role during Luteolysis in Women

Rachel E. Dickinson, Michelle Myers and W. Colin Duncan

Medical Research Council Human Reproductive Sciences Unit (R.E.D.), Centre for Reproductive Biology, and Obstetrics and Gynaecology (R.E.D., M.M., W.C.D.), Department of Reproductive and Developmental Sciences, Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom

Address all correspondence and requests for reprints to: Rachel E. Dickinson, Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: r.dickinson{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human corpus luteum (CL) undergoes luteolysis, associated with marked tissue and vascular remodeling, unless conception occurs and the gland is rescued by human chorionic gonadotropin (hCG). In Drosophila the Slit gene product, a secreted glycoprotein, acts as a ligand for the roundabout (robo) transmembrane receptor. Together they influence the guidance and migration of neuronal and nonneuronal cells. In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified. ROBO1, SLIT2, and SLIT3 are also inactivated in human cancers and may regulate apoptosis and metastasis. Because processes such as apoptosis and tissue remodeling occur during the regression of the CL, the aim of this study was to investigate the expression, regulation, and effects of the SLIT and ROBO genes in human luteal cells. Immunohistochemistry and RT-PCR revealed that SLIT2, SLIT3, ROBO1, and ROBO2 are expressed in luteal steroidogenic cells and fibroblast-like cells of the human CL. Furthermore, using real-time quantitative PCR, expression of SLIT2, SLIT3, and ROBO2 was maximal in the late-luteal phase and significantly reduced after luteal rescue in vivo with exogenous hCG (P < 0.05). Additionally, hCG significantly inhibited SLIT2, SLIT3, and ROBO2 expression in cultured luteinized granulosa cells (P < 0.05). Blocking SLIT-ROBO activity increased migration and significantly decreased levels of apoptosis in primary cultures of luteal cells (P < 0.05). Overall, these results suggest the SLIT/ROBO pathway could play an important role in luteolysis in women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN CORPUS luteum (CL) is a highly vascular endocrine gland, measuring up to 2 cm in diameter, that forms during luteinization when the granulosa cells of the postovulatory follicle undergo terminal differentiation. During its short life span, it undergoes intense tissue and vascular remodeling. In a nonconception cycle, around 10–12 d after ovulation, progesterone levels fall and the CL undergoes structural and functional regression (1). The molecular mechanisms and regulation of luteolysis in women is not yet clear, but it involves tissue remodeling with an increase in apoptosis (2), expression of connective tissue growth factor (3), matrix metalloproteinase (MMP) activity (4). and the influx of macrophages (5). In early pregnancy, human chorionic gonadotropin (hCG) acts through the LH receptor to rescue the CL from regression and maintain its structural and functional integrity (6).

The SLIT/roundabout (ROBO) pathway has roles in tissue growth, development, and remodeling. In Drosophila the Slit gene product acts as a ligand for the robo transmembrane receptor (7, 8). In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified (8, 9, 10, 11). Because the Slits are secreted glycoproteins they have the potential to be local paracrine and autocrine regulators of cell function. It has been well documented that the Slit/Robo interaction plays a pivotal evolutionary conserved role in guiding axons during the assembly of the nervous system by acting as a repulsive cue (12).

With the exception of Slit1, past research implies the Slit and Robo proteins are also expressed in a variety of nonneuronal tissues (13, 14, 15). Indeed Robo4 is an unusual member of the Robo family because it appears to be specifically expressed in the vasculature system in which it inhibits angiogenesis (16, 17, 18). Furthermore, homozygous knockout mice with deletions in a certain Slit or Robo often die at birth and suffer from severe developmental abnormalities in their lungs, diaphragm, heart, or kidneys (19, 20, 21, 22).

In addition to a role in organ development, there is evidence for a role in somatic cell function. Slit2 hinders chemotaxis of vascular smooth muscle cells and leukocytes as well as axons (23, 24, 25). Slit2 may also have an important antiinflammatory role in vivo as treatment with the protein improved the renal function and disease histology of rats with crescentic glomerulonephritis (26).

There is mounting evidence to suggest that the Slit/Robo interaction may play an important role in suppressing tumorigenesis by promoting apoptosis and inhibiting cell migration (27, 28). Previous research implied ROBO1 (3p12) and SLIT2 (4p15.2) are inactivated through deletions and hypermethylation of their promoter regions in a number of tumor types including breast, lung (29, 30), and cervical cancer (31, 32, 33, 34). In addition, SLIT3 (5q34–35) and, to a lesser extent, SLIT1 (10q23.3–24) are inactivated through promoter region hypermethylation or loss of heterozygosity in human cancers (35), including ovarian germ line tumors (36).

Reexpression of SLIT2 suppressed breast tumor and glioma cell growth, induced apoptosis in colorectal and lung cancer cells (29, 37, 38, 39), and inhibited the invasion of breast tumor and medulloblastoma cells (40, 41). Furthermore, Robo1+/– mice had a 3-fold increased likelihood of developing spontaneous lung carcinomas, compared with wild-type mice (42). With such clear roles in cell growth, apoptosis, and tissue and vascular remodeling in physiological and pathological cell systems, the Robos and Slits are excellent candidates as novel paracrine regulators of the CL.

There is, however, no evidence in literature linking the SLITs and ROBOs to ovarian physiology. We hypothesized that the SLIT/ROBO pathway was involved in regulating the marked tissue remodeling in the human CL. We therefore investigated the expression, localization, regulation, and action of the SLIT and ROBO genes in the human CL and primary cultures of steroidogenic and luteal fibroblast-like cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of human tissue
Tissue collection was approved by the local medical research ethics committee, and all women gave informed consent. Human CLs (n = 13) were enucleated at the time of surgery from women with regular menstrual cycles undergoing hysterectomy for benign conditions and dated on the basis of the urinary LH surge as described previously (6). In this study, four CLs were classified as early luteal (LH+1 to LH+5), four as midluteal (LH+6 to LH+10), and five as late luteal (LH+11 to LH+14).

At operation the CL was quartered to ensure that each quarter contained all cellular elements, fixed in 10% neutral buffered formalin for subsequent immunohistochemistry, and snap frozen for mRNA extraction. In addition, frozen tissue quarters were also available from four CLs that had been rescued (daily doubling doses of exogenous hCG from LH+7 for 5–8 d) as described previously (4). Human fetal ovary samples were ethically approved and total RNA from these tissues was kindly provided by Professor R. A. Anderson (Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK) (43).

Isolation of primary cells
The medical ethics committee separately approved the collection of cells from patients undergoing assisted conception. With patient consent, follicular fluid was collected from women undergoing transvaginal oocyte retrieval for in vitro fertilization after ovarian stimulation using a standard procedure (44). Isolation of luteinized granulosa cells (LGCs) was carried out as described previously (3). Fibroblast-like cells were obtained from prolonged cultures of follicular aspirates as described previously and identified morphologically (3). We had previously characterized the cellular composition of primary cell cultures from follicular fluid enabling us to identify the different cellular types with confidence (3). Briefly, medium was changed weekly and after 4 wk the fibroblast-like cells had reached confluence and the luteinized granulosa cells had disappeared. Cells were passaged twice before being seeded for experimental procedures described below. Endothelial enriched cultures were obtained from follicular aspirates by following a protocol described previously (45). Normal human myometrial uterine microvascular endothelial cells were bought commercially (Clonetics, Lonza Wokingham Ltd., Berkshire, UK). Primary human umbilical vein endothelial cells were isolated from umbilical cords and cultured as described previously with ethical approval (46).

Derivation of fibroblast-like cells from human corpora lutea
Cell suspensions were prepared from freshly collected CLs and placed in flasks containing DMEM/F12 Ham (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) as described previously (3). Fibroblast-like cells were identified morphologically. Briefly, medium was changed weekly and after 4 wk the fibroblast-like cells had reached confluence and the luteinized granulosa cells had disappeared. Cultures were passaged twice before being seeded for experimental procedures described below.

Primary cell culture
To assess the acute effects of hCG, activin A, and cortisol on steroidogenic cells, pooled luteinized granulosa cells (1–1.5 x 105 cells/well of three to five patients) were cultured in 24-well plates precoated with matrigel (BD Biosciences, Bedford MA) in serum-free medium (insulin transferrin-sodium selenite supplemented DMEM/F12 Ham mixture; Life Technologies). Medium was changed every 2–3 d over the course of the culture period. After 6–8 d in culture, cells were treated with recombinant hCG (Serono Laboratories, Welwyn Garden City, UK; 100 ng/ml) in conjunction with low-density lipoprotein (LDL; Sigma-Aldrich Corp., Gillingham, UK; 50 mg/liter), recombinant human activin A (R&D Systems, Inc., Abingdon, UK; 25 ng/ml), or cortisol (100 nM). For each treatment controls, consisting of an equivalent volume of the carrier solution to the concentration added, were conducted in parallel. After 24 h RNA was extracted from the cells using the RNeasy minikit (QIAGEN Ltd., Crawley, UK) according to the manufacturer’s instructions. All RNA was deoxyribonuclease treated with column deoxyribonuclease I (QIAGEN). Each experiment was carried out at least three times to reduce the possible effects of biological variability.

To mimic the luteal phase in primary cell culture, luteinized granulosa cells were plated as described above and maintained for 12 d as described previously (44). Briefly, cells were stimulated with 1 ng/ml hCG and 50 mg/liter LDL from d 2 of culture, and this treatment was repeated every second day until d 7. At this stage the remaining cells were given either 50 mg/liter LDL alone or 100 ng/ml hCG and 50 mg/liter LDL for an additional 6 d. Total RNA was extracted from cells as described above at d 3 and d 7 along with d 13 in the absence or presence of hCG. To confirm that the progesterone secretion in these samples mirrored the profile of an in vivo luteal phase, media were also collected at the above time points and measured for progesterone levels using an in-house RIA as previously reported (44).

Cultures of luteal fibroblast-like cells were derived as described above and transferred to 24-well plates (6–8 x 104 cells/well) to investigate the acute effect of cortisol on these cells. After 6 h under serum-free conditions, the medium was replaced with medium containing either cortisol (100 nM) or an equivalent volume of carrier solution. After 24 h total RNA was extracted from the cells as described above.

Wound-healing assay
Fibroblast-like cells were seeded at 1 x 105 cells/well in 12-well plates, resulting in a confluent monolayer. After 24 h DMEM/F12 Ham supplemented with 10% FBS was replaced with serum-free media containing recombinant rat ROBO1/Fc chimera (R&D Systems; 1 µg/ml) or the equivalent volume of PBS/0.1% BSA. The cells were maintained in serum-free media prior and during the experiment to exclude the possibility of cell growth masking the effects of migration. After a further 24 h, each well of cells was scratched with a sterile 200 µl pipette tip. The extent of wound healing was observed and images were captured at 0 and 24 h after the scratch was made using a Leitz Labovert inverted microscope (Ernst Leitz GmbH Wetzlar, Germany) equipped with a Leica DFC280 digital camera (Leica Microsystems UK Ltd., Milton Keynes, UK). The experiment was carried out four times to reduce the possible effects of biological variability.

For quantification, each image was inserted into a Microsoft Office PowerPoint document (Richmond, CA), and the wound width was measured at three different points. A mean wound width was then calculated for each image. The overall relative mean wound size at 0 h for control and ROBO1/Fc-treated cells was assigned a value of 100%. The overall relative mean wound size at 24 h for control and ROBO1/Fc-treated cells was then expressed as a percentage of the 0 h value.

Caspase-3 and -7 activity assay
To measure caspase-3 and -7 activities in fibroblast-like cells and luteinized granulosa cells, the Caspase-Glo 3/7 assay was performed according to the manufacturer’s instructions (Promega UK Ltd., Southampton, UK). Fibroblast-like cells or luteinized granulosa cells were seeded at 2 x 104 cells/well in 96-well plates. After 24–72 h fresh DMEM/F12 Ham supplemented with 10% FBS was added containing recombinant rat ROBO1/Fc chimera (R&D Systems; 1 µg/ml) or the equivalent volume of PBS/0.1% BSA. Treatments were carried out in technical quadruplicate. DMEM/F12 Ham supplemented with 10% FBS containing recombinant human TNF-{alpha} (R&D Systems; 2 ng/ml) was added to another two wells and served as a positive control for the assay. TNF-{alpha} was chosen because previous research has suggested that it is expressed and capable of inducing apoptosis in the human CL (47, 48). Because serum can generate a background caspase activity signal, an additional two wells contained cell culture medium and carrier solution without any cells.

After 48 h Caspase-Glo 3/7 reagent was added directly to the cells in culture medium in a 1:1 ratio. The well contents were then mixed and incubated at room temperature for up to 3 h. This resulted in cell lysis, followed by caspase cleavage of the substrate and generation of a glow-type luminescent signal, produced by luciferase. Luminescence was measured using a FLUOstar OPTIMA microplate reader (BMG Labtech Ltd., Aylesbury, UK). Luminescence was directly proportional to the amount of caspase activity present. The value for the no cell control was subtracted from the experimental values. The experiment was carried out three to four times to reduce the possible effects of biological variability.

Expression analysis
Extracted RNA from frozen human CLs and cultured cells was used as a template for cDNA synthesis using Taqman reverse transcriptase reagents (Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. Primers used for PCR were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) from DNA sequences obtained at Ensembl Genome Browser (http://www.ensembl.org/index.html). To minimize the amplification of genomic DNA, the forward and reverse primers for each gene were specific for different exons. Primers were synthesized by MWG-AG Biotech (Milton Keynes, UK) and are described in Table 1Go. The PCR thermocycle consisted of an initial denaturation of 5 min at 95 C followed by 35 cycles of 95 C for 30 sec, annealing temperature for 30 sec, 72 C for 30 sec, and a final extension of 10 min at 72 C. PCR products were visualized on a 2% agarose gel with added ethidium bromide.


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  		TABLE 1. Primers used in the expression analysis of candidate genes

 
Real-time quantitative PCR
RNA was extracted and reverse transcribed as described above. A standard curve was also generated with serial dilutions of cDNA synthesized from human fetal brain total RNA (Stratagene Europe, Amsterdam, The Netherlands). Real-time PCR amplification was then performed in duplicate 10-µl reactions using Platinum SYBR Green quantitative PCR SuperMIX-UDG (Invitrogen Ltd., Paisley, UK) following the manufacturer’s instructions and using the ABI 7900HT fast real-time PCR system instrument (Applied Biosystems). Primers used were the same as for the expression analysis and are described in Table 1Go. The cycling program consisted of an initial UDG incubation of 2 min at 50 C and then a denaturation step for 2 min at 95 C. This was followed by 45 cycles of 95 C for 15 sec, 60 C for 10 sec, and 72 C for 30 sec. The default setting for the ABI instrument was used for the melting curve analysis.

The ABI analysis software calculated quantitative values for each sample by comparing the sample threshold cycle number, where the increase in the signal associated with exponential growth of PCR products begins to be detected, to the standard curve, according to the manufacturer’s manuals. In all cases the level of gene expression within the samples lay within the boundaries of the corresponding standard curve. Because the precise quality and amount of cDNA that was added to each reaction mix was difficult to assess, transcripts of glucose-6-phosphate dehydrogenase (G6PDH), a housekeeping gene, were also quantified for each sample as described above. This gene is not regulated in the samples under investigation and therefore acted as an endogenous control. Each sample was normalized on the basis of its G6PDH content by dividing the amount of target gene by the amount of housekeeping gene.

Immunohistochemistry
To investigate the localization of ROBO1 and SLIT2 proteins in human tissues, 5-µm paraffin tissue sections of human CL prepared on poly-L-lysine-coated microscope slides were examined. Goat polyclonal antibodies raised against amino acids 19–560 of rat ROBO1 (AF1749; R&D Systems) and an internal region of human SLIT2 (sc-16619; Santa Cruz Biotechnology, Santa Cruz, CA) were used for detection. First, sections were dewaxed, rehydrated, washed in PBS, subjected to pressure cooker antigen retrieval in 0.01 M citric acid (pH 6.0) for 5 min, and left to cool to room temperature. Next, all sections were washed and placed in 3% H2O2/double-distilled H2O for 10 min to block any endogenous peroxidase activity. This was followed by an avidin and biotin block and a further block using normal rabbit serum (NRS; Diagnostics Scotland, Edinburgh, UK) diluted 1:5 in Tris-buffered saline for 1 h at room temperature. Sections were then incubated with SLIT2 or ROBO1 antibodies and diluted to a final concentration of 20 µg/ml and 10 µg/ml, respectively, in NRS, for 16–24 h at 4 C. This was followed by washes with PBS on an orbital shaker. Sections were then incubated at room temperature for 1 h with biotinylated rabbit antigoat IgG (Vector Laboratories Inc., Burlingame, CA) diluted to a final concentration of 3 µg/ml in NRS. After washing, as before, the sections were incubated in avidin-biotin complex-horseradish peroxidase (Dako UK Ltd., Ely, UK) for 1 h.

Binding was visualized by subsequent incubation with liquid 3,3'-diaminobenzidine tetra-hydrochloride (Dako UK). Sections were counterstained lightly with hematoxylin to enable cell identification. Negative controls were performed in parallel using an identical protocol except primary antibody was replaced with blocking serum containing nonspecific immunoglobulins at the same concentration. Images were captured using a Provis microscope (Olympus Corp. Optical Co., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak Co., Rochester, NY), stored on a Hewlett-Packard computer and assembled using Photoshop 7.0.1 (Adobe, Mountain View, CA).

Statistical analysis
Statistical analyses were carried out after confirmation of normal distributions for parametric analysis, using a paired t test when treatment and control samples were analyzed or with a one-way ANOVA when more than two groups were compared. When group means were significantly different by ANOVA (P < 0.05), pairwise comparisons were performed using Bonferroni’s multiple comparisons test. All statistical tests are highlighted in the figure legends, and differences are given (*, P < 0.05; **, P < 0.01; or ***, P < 0.001). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Members of the SLIT and ROBO families are expressed in the human CL
To investigate the expression of all three SLIT and four ROBO genes in the human ovary, primers specific for all family members were designed and PCR products were sequenced to confirm their identity. The relative abundance of each gene transcript was examined in human fetal ovary and adult CL using RT-PCR (Fig. 1Go). All members of the SLIT and ROBO family were detected in the human fetal ovary, which supports their role in organogenesis (Fig. 1Go). However SLIT2, SLIT3, ROBO1, ROBO2, and ROBO4 were also expressed in the mature adult CL (Fig. 1Go).


Figure 1
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  		FIG. 1. Expression analysis of the SLIT and ROBO gene families. RNA extracted from human fetal ovary (19 wk), midluteal phase CL (mid CL), cultured luteinized granulosa cells (LGC), luteal fibroblast-like cells, and endothelial enriched cultures was used as a template for RT-PCR. A, All members of the SLIT family were expressed in the fetal ovary. The CL, cultured LGC, and cultured luteal fibroblast-like cells also expressed SLIT2 and SLIT3. SLIT2 was also expressed in endothelial cell cultures. B, Similarly, all the ROBO genes were expressed in the human fetal ovary. ROBO1, ROBO2, and ROBO4 were also expressed in the adult CL. ROBO1 and ROBO2 were expressed in cultured LGC, endothelial enriched cultures, and cultured luteal fibroblast-like cells. ROBO4 seemed to be endothelial cell specific. Control samples contained PCR master mix without any DNA (–). Microvascular endothelial cells (MVECs) and human umbilical vascular endothelial cells (HUVECs) were also included as controls. The housekeeping gene G6PDH was used as an internal control to ensure RNA integrity and equal loading.

 
To assess which cell types express these genes their expression was examined in primary cell cultures of different luteal cells and endothelial cells. ROBO1 and ROBO2 seemed to be expressed in luteinized granulosa cells and luteal fibroblast-like cells, although, as expected, ROBO4 was endothelial cell specific (Fig. 1Go). Their ligands, SLIT2 and SLIT3, were also expressed by luteal fibroblast-like cells and luteinized granulosa cells.

This localization was confirmed by immunohistochemistry for SLIT2 and ROBO1 in the CL (Fig. 2Go). Mid-luteal sections showed some positive ROBO1 staining but very weak SLIT2 expression (Fig. 2Go, A and B). Late-luteal sections showed more intense ROBO1 and SLIT2 staining with both proteins localized to the cytoplasm of granulosa-lutein, thecal-lutein and luteal fibroblasts (Fig. 2Go, C and D). No staining could be detected in negative control sections (Fig. 2EGo). The most marked immunostaining for ROBO1 and SLIT2 was observed in very late luteal CLs, suggesting that the expression of SLIT2 in particular may vary across the luteal phase (Fig. 2Go, F and G).


Figure 2
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  		FIG. 2. SLIT2 and ROBO1 are localized to luteal steroidogenic cells and fibroblast-like cells in the human CL. A, Light-field microscopy of a mid-luteal human CL from LH+6 showing weak positive brown DAB staining for ROBO1 in the granulosa-lutein cells (GLC), theca-lutein cells (TLC), fibroblast-like cells (F) and blood vessels (BV). B, Very weak brown diaminobenzidine (DAB) staining for SLIT2 in GLC, TLC, fibroblasts, and blood vessels of a midluteal CL. C, Light-field microscopy of late-luteal human CL from LH+12 showing positive brown DAB staining for ROBO1 in the GLC, TLC, and some staining in the fibroblast-like cells (F). D, Positive brown DAB staining for SLIT2 in GLC, TLC, and fibroblast-like cells (F) of a late-luteal (LH+12) CL. E, No DAB staining was observed in the negative control serial section. F, Brown DAB staining for ROBO1. G, Most markedly, SLIT2 was seen in the GLC, TLC, and fibroblast-like cells (F) of a very late (LH+14) CL. Pictures were taken with x20 magnification. All the scale bars represent 100 µm.

 
As the corpus luteum ages, there is a significant increase in the expression of SLIT2, SLIT3, and ROBO2, which is repressed by hCG treatment
To assess whether expression of SLIT2, SLIT3, ROBO1, and ROBO2 was differentially regulated, their expression was examined across the luteal phase in well-characterized CL in the absence and presence of hCG (Fig. 3Go). Whereas ROBO1 expression did not change notably over the luteal phase, as the CL aged, there was a significant increase in the levels of SLIT2, SLIT, and ROBO2 (P < 0.01; P < 0.001, and P < 0.01, respectively, ANOVA) (Fig. 3Go, A–D). SLIT2 expression was significantly different in mid- vs. late luteal CL (P < 0.05, Bonferroni’s multiple comparison test). SLIT3 and ROBO2 expression was significantly different between early and late and mid- and late-luteal phases (P < 0.001 and P < 0.01, respectively, Bonferroni’s multiple comparison test).


Figure 3
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  		FIG. 3. Expression of SLIT and ROBO genes in staged human corpora lutea using real-time quantitative PCR. A, SLIT2 expression changed over the luteal phase (P < 0.01, ANOVA). SLIT2 expression increased from the mid-luteal phase to the late-luteal phase (P < 0.05, Bonferroni), and luteal rescue by hCG reduced SLIT2 expression (P < 0.05, Bonferroni). B, Likewise, expression of SLIT3 changed in the different CL samples (P < 0.001, ANOVA). There was higher SLIT3 expression in the late-luteal samples than the early and mid-luteal samples (P < 0.001, Bonferroni) and after luteal rescue with exogenous hCG (P < 0.05, Bonferroni). C, ROBO1 expression remained relatively unchanged across the luteal phase and after luteal rescue with exogenous hCG. D, ROBO2 expression did change, however, across the luteal phase (P < 0.01, ANOVA). ROBO2 expression was higher in the late than the early and midluteal samples (P < 0.01, Bonferroni) and also higher than CLs rescued with exogenous hCG (P < 0.05, Bonferroni). *, P < 0.05; **, P < 0.01; or ***, P < 0.001. Differences were considered significant at P < 0.05.

 
Expression of SLIT2, SLIT3, and ROBO2 reached a maximum in the late-luteal phase when the CL was approaching luteolysis. In addition, expression of SLIT2, SLIT3, and ROBO2 was significantly reduced after luteal rescue in vivo with exogenous hCG (P < 0.05, Bonferroni’s multiple comparison test). These results suggest that expression of the SLIT/ROBO pathway is regulated and maximal during luteolysis.

Chronic manipulation of hCG in cultures of human luteinized granulosa cells causes a significant reduction in SLIT2, SLIT3, and ROBO2 expression
The CL contains many different cell types, which may change in proportion across the luteal phase. To investigate the direct effect of hCG on SLIT and ROBO2 expression in luteal steroidogenic cells, primary cultures of luteinized granulosa cells were studied using an in vitro model designed to mimic the stages of the luteal phase (Fig. 4Go). After 7 d in culture with low concentrations of hCG, the hormone was either increased to simulate luteal rescue or removed to mimic luteolysis. Using real-time quantitative PCR, SLIT2, SLIT3, and ROBO2 expression changed significantly (P < 0.01, P < 0.01, and P < 0.05, respectively, ANOVA). Expression levels of all three genes was higher when hCG treatment was withdrawn (P < 0.05, Bonferroni’s multiple comparison test). Furthermore, the presence of increased hCG caused a reduction in the expression of these three genes supporting the findings seen in the dated CL samples (P < 0.05, Bonferroni’s multiple comparison test) (Fig. 4Go, A–C).


Figure 4
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  		FIG. 4. SLIT2, SLIT3, and ROBO2 expression changed significantly in cultures of luteinized granulosa cells designed to mimic the luteal phase. Cells were given low-dose hCG stimulation for 1 wk, and RNA was extracted at d 3 and d 7. At d 7 cells were then cultured for a further 6 d in the complete absence (–), or with a maximal dose (+), of hCG, and RNA was extracted from these cells at d 13. A, SLIT2 expression changed in this model (P < 0.01, ANOVA). Withdrawal of hCG led to an increase in SLIT2 expression (P < 0.05, Bonferroni). B, Expression of SLIT3 similarly changed (P < 0.01, ANOVA) with expression highest when hCG was withdrawn from the cultures (P < 0.05, Bonferroni). C, ROBO2 expression showed the same pattern (P < 0.05, ANOVA). Absence of hCG lead to increased ROBO2 expression (P < 0.05, Bonferroni). *, P < 0.05; **, P < 0.01; or ***, P < 0.001. Differences were considered significant at P < 0.05.

 
Expression of SLIT2 and SLIT3 is acutely regulated by cortisol and hCG in primary cell cultures
To determine whether the expression of SLIT2, SLIT3, ROBO1, and ROBO2 could be acutely regulated by hCG, its effect on primary cultures of luteinized granulosa cells was examined (Fig. 5AGo). Treatment with hCG for 24 h significantly reduced SLIT2, SLIT3, and ROBO2 expression (P < 0.01, P < 0.05, and P < 0.05, respectively, paired t test). In addition, treatment with LH for 24 h also significantly reduced mRNA levels of SLIT2 and ROBO2 (data not shown).


Figure 5
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  		FIG. 5. Cortisol and hCG negatively regulate SLIT2 and SLIT3 expression in short-term primary cell cultures. The mean expression of control-treated cells was normalized to 1. Values are the mean ± SEM of three independent experiments. A, Culturing LGC with hCG (100 ng/ml) for 24 h significantly reduced SLIT2, SLIT3, and ROBO2 (P < 0.01, P < 0.05, and P < 0.05, respectively, paired t test) but not ROBO1 expression. B, SLIT2 and SLIT3 expression was also reduced in LGC treated with cortisol (100 nM) for 24 h (P < 0.05, paired t test). ROBO2 expression remained relatively unchanged. C, Likewise luteal fibroblast-like cells treated with 100 nM cortisol, for 24 h, had significantly diminished SLIT2 and SLIT3 expression (P < 0.05, paired t test). Cortisol treatment did not significantly affect ROBO2 expression. *, P < 0.05; **, P < 0.01; or ***, P < 0.001. Differences were considered significant at P < 0.05.

 
As we have shown that in the human CL luteal rescue with hCG is associated with increased local generation of the glucocorticoids by 11β-hydroxysteroid dehydrogenase (11βHSD) type 1 (49), we investigated whether cortisol could also acutely modulate the expression of SLIT2, SLIT3, and ROBO2. Cortisol significantly diminished expression of SLIT2 and SLIT3 in primary cultures of luteinized granulosa cells (P < 0.05, paired t test) (Fig. 5BGo). SLIT2 and SLIT3 expression was also significantly reduced in cortisol-treated luteal fibroblast-like cells (P < 0.05, paired t test) (Fig. 5CGo). Cortisol did not significantly effect ROBO2 expression in either cell type.

Because activin A has opposing actions to hCG and cortisol in the human CL (50), we assessed whether activin could regulate expression of SLIT2 and SLIT3 in primary cultures of luteinized granulosa cells (data not shown). The expression of SLIT2 and SLIT3 was not significantly effected by treatment with activin A. In addition, treating luteal fibroblast-like cells with activin A did not significantly change SLIT2 or SLIT3 expression (data not shown).

Blocking SLIT activity increases cell motility and decreases apoptosis in luteal cells
Because our findings imply the SLIT-ROBO pathway could play a role in the regression of the CL, we investigated the effect of inhibiting SLITs on the function of luteal fibroblast-like cells and luteinized granulosa cells. Cells were treated with a recombinant ROBO1/Fc chimera that acts as a ligand trap to inhibit SLIT/ROBO signaling. Because this pathway inhibits migration of different somatic cells (51, 52), the migration of luteal fibroblast-like cells was studied using a wound healing assay. Because luteinized granulosa cells do not migrate, they were not suitable for this study. Twenty-four hours after a scratch had been made, ROBO1/Fc chimera-treated cells had invaded the wound, whereas control cells did not noticeably move (Fig. 6AGo). Treatment with the ROBO1/Fc chimera caused a significant 52% reduction in the width of the wound after 24 h (P < 0.01, paired t test). Conversely, the wound stayed relatively the same width in control-treated cells (P > 0.05, paired t test) (Fig. 6BGo). Cells were cultured in serum-free media prior and during this experiment to exclude the possibility of proliferation masking the effects of migration.


Figure 6
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  		FIG. 6. Inhibiting SLIT activity increases cell migration. A, Luteal fibroblast-like cells were treated with the ROBO1/Fc chimera or PBS/0.1% BSA (control) under serum-free conditions. After 24 h, an artificial wound was scratched through them (0 h), and 24 h later, ROBO1/Fc treated cells had invaded the wound in all four independent experiments, whereas no invasion occurred in the control cells. Images were captured with x4 magnification. B, Images taken of the scratched control and ROBO1/Fc-treated cells at 0 and 24 h were inserted into a Microsoft PowerPoint document and the width of the wound was measured. The overall relative mean wound size at 0 h for control and ROBO1/Fc-treated cells was set at 100%. Values are the mean ± SEM of four independent experiments. After 24 h, the wound width in ROBO1/Fc-treated cells was significantly reduced by 52% (P < 0.01, paired t test). In contrast, the wound width in control cells remained relatively unchanged over 24 h (P > 0.05, paired t test). *, P < 0.05; **, P < 0.01; or ***, P < 0.001. Differences were considered significant at P < 0.05.

 
Because SLIT2 can induce apoptosis (29, 38) associated with activation of caspases, caspase-3 and -7 activity was measured in luteal fibroblast-like cells and luteinized granulosa cells treated with and without the ROBO1/Fc chimera. Treatment with the ROBO1/Fc chimera caused a significant 20% reduction in activation of caspase-3 and -7 in luteal fibroblast-like cells and luteinized granulosa cells (P < 0.0001 and P < 0.05, respectively, paired t test) (Fig. 7Go, A and B).


Figure 7
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  		FIG. 7. Blocking SLIT-ROBO signaling decreases caspase-dependent apoptosis. A and B, Forty-eight hours after treatment with either the ROBO1/Fc chimera or PBS/0.1% BSA (control), the activity of caspase-3 and -7 was measured using the Caspase-Glo 3/7 assay (Promega). The mean luminescence measured in control-treated cells was normalized to 1. Values are the mean ± SEM. Significantly less caspase activity was observed in the ROBO1/Fc-treated fibroblast-like cells (n = 4) (P < 0.0001, paired t test) (A). Similarly, LGCs treated with the ROBO1/Fc chimera also contained significantly less caspase activity (n = 3) (P < 0.05, paired t test) (B). *, P < 0.05; **, P < 0.01; or ***, P < 0.001. Differences were considered significant at P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Every month, during a 2-wk period, the CL forms and undergoes regression through the tight regulation of cell proliferation, apoptosis, differentiation, angiogenesis, and tissue remodeling. If a pregnancy occurs, hCG acts to maintain the CL’s structural and functional integrity (6). The molecular and cellular mechanisms involved in regression and maintenance of the CL, however, are still not fully understood. Although the SLITs and their ROBO receptors were only recently discovered, there is mounting evidence to suggest that they promote apoptosis and inhibit cell migration, whereas their activities are also perturbed in a variety of cancers (27).

Because these processes are also involved in luteolysis, the aim of this study was to investigate, for the first time, whether the SLIT-ROBO pathway also plays a role in human ovarian physiology. The results implied that the adult CL expresses, at the RNA level, most members of the SLIT and ROBO families. As the life span of the CL progresses, there also seems to be a significant increase in the expression of SLIT2, SLIT3, and ROBO2. In addition, treating CL or cultured luteinized granulosa cells from the luteinizing follicle with hCG, imitating the effects of early pregnancy, caused a significant reduction in SLIT2, SLIT3, and ROBO2. Furthermore, inhibiting SLIT-ROBO signaling led to increased motility and reduced apoptosis in cultured luteal cells. Overall, these results suggest a novel role for the SLIT-ROBO pathway in the ovary and in particular the luteolytic process.

There is no evidence in the literature of a role for the SLIT-ROBO pathway in normal ovarian physiology in women or other mammalian species. Indeed, although knockout mice have been generated (19, 20, 21, 22), the lethal effect of the knockout means that ovarian physiology in these mice has not been studied. Investigations using conditional knockout models would be valuable but have not been reported thus far.

A key finding from this research was the significant changes in SLIT2, SLIT3, and ROBO2 mRNA expression over the luteal phase. There was a significant increase in expression of all three genes in the late-luteal staged CLs. These findings support the theory that SLIT-ROBO signaling is increasing in the lead-up to CL regression and could have an important function in luteolysis. It is interesting that both the SLIT ligands and their ROBO receptor seem to be similarly differentially regulated at the same time. Such similar regulation of ligand and receptors has been described in the CL. Vascular endothelial growth factor and its receptors showed the same patterns of expression in the porcine CL in which levels were significantly higher in the early and mid-luteal phases (53). Additionally, in the primate ovary, dynamic expression and regulation of the CRH/urocortin-receptor-binding protein system was observed with both ligands and receptor levels showing maximal expression in the early and midluteal phases before declining (54). Such a pattern of parallel ligand and receptor regulation implies that regulation of the pathway may have functional importance.

It is worth noting that these studies were all performed using real-time quantitative PCR, so it is not known whether expression of the SLITs and ROBOs could also be post translationally modified over the luteal phase. Our immunohistochemistry data did suggest that SLIT2 and ROBO1 proteins are produced in the human CL, and they seemed to be expressed at higher levels in the regressing CL. Unfortunately, we did not have enough tissue available to test this fully by Western blotting. However, the localization of immunostaining mirrored the primary cell PCR expression, and in the nervous system, SLIT2 mRNA and protein expression also change in parallel (55).

Another interesting discovery was the possible hormonal regulation of the SLIT-ROBO pathway by hCG. CL tissue from women who had been given daily doubling doses of exogenous hCG from the mid-luteal phase for 5–8 d had significantly reduced expression of SLIT2, SLIT3, and ROBO2. This would suggest that during early maternal recognition of pregnancy, hCG acts on the LGC to inhibit the SLIT-ROBO pathway. Prolonged cultures of luteinized granulosa cells, in which the luteal phase had been mimicked, mirrored these findings. A significant increase in SLIT2, SLIT3, and ROBO2 expression was seen when hCG was withdrawn, in marked contrast to when hCG concentrations were increased. We also found that hCG had an acute negative effect on SLIT2, SLIT3, and ROBO2 expression in cultures of luteinized granulosa cells. This provides evidence of physiological G protein-coupled ligand-induced regulation of the SLIT-ROBO pathway.

During the formation and demise of the CL, there are profound changes in the extracellular matrix that allows for alterations in cell migration, vascularization, and fibrosis (6). Tissue remodeling during luteolysis has been associated with increased activin A activity (50). Activin A has effects on tissue fibroblast function. It can stimulate connective tissue growth factor expression, which has been implicated in the fibrosis and remodeling associated with luteolysis (3). In addition, it increases stromal cell MMP-2 activity, an enzyme that plays a key role in the breakdown of the extracellular matrix (50). We therefore hypothesized that activin A would regulate expression of the luteal SLIT-ROBO system. However, in this study we did not see a significant effect by activin A on SLIT2 or SLIT3 expression.

Slits, however, may have a role in luteal fibroblast function as blocking SLIT-ROBO activity caused cells to invade the scratch using a wound-healing assay. This suggested the SLIT-ROBO pathway may inhibit cell migration. Serum-free media were used during this assay to reduce cell growth; however, we cannot dismiss the possible effect ROBO1 signaling may have directly on fibroblast proliferation. Therefore, it is possible that there is increased fibroblast proliferation in response to ROBO1/Fc treatment. The potential cell motility effect comes as no surprise because the majority of previous studies in other cellular systems have implied that SLIT-ROBO signaling has a repulsive effect on migration (52). It is generally thought that the SLIT-ROBO interaction mediates its effect through signal transduction pathways that include Enabled, GTPase-activating proteins, and inactivation of RhoGTPases including Cdc42. Consequently, this leads to a decrease in actin polymerization (56, 57). In breast cancer cells, SLIT2 is thought to block stromal-derived factor-1-mediated chemotaxis and chemoinvasion partly by influencing the secretion of MMP-2 and -9 (40).

Luteolysis is also known to be associated with vascular regression (6). Interestingly, the SLIT-ROBO pathway has also been implicated in angiogenesis. Past studies have found that ROBO4 shows highly endothelial cell-specific expression in vitro and in vivo (51). Our data would suggest that this is also the case in the human adult CL. The actual signaling mechanism and the effect of this system on endothelial cells have been the subject of some controversy. Interaction between Slit2 and Robo4 has been reported in one study (16), whereas another was unable to detect Slit-Robo4 binding (17). Most studies have suggested that Slit2 inhibits endothelial cell migration (16, 25). It is not known, however, whether, as well as inhibiting migration, the SLIT-ROBO system can promote apoptosis of endothelial cells in vivo.

There is an increase in cell death in the human CL during the late-luteal phase, and structural luteolysis is thought to involve programmed cell death. Possible factors mediating human luteal cell apoptosis are the Fas and Fas-ligand system, Bcl-2 family, TNF-{alpha}, and caspase-3 (58). In fact, a protein kinase C inhibitor, staurosporine, induced apoptosis with increases in both caspase-3 and -9 activities in cultured human luteinized granulosa cells (59). We found a significant decrease in casapase-3 and -7 activities in luteal fibroblast-like cells and luteinized granulosa cells in which SLIT-ROBO activity had been blocked. Past findings have implied SLIT2 can induce apoptosis in colorectal tumor cells and increase breast cancer cell death (29, 38). Previous research has also suggested that SLIT2 promotes activation of caspase-3 and thus apoptosis in lung tumor cells (39).

The mechanism by which SLIT2 exerts this effect is unclear; however, it may involve the deleted in colorectal cancer (DCC) pathway. Binding of SLIT2 promotes an interaction between ROBO1 and DCC (60). This may cause DCC to disassociate from Netrin-1, its ligand, thereby activating proapoptotic pathways through caspase-3 and -9 (61). Previous research demonstrated DCC and Netrin-1 are expressed in the ovary. Furthermore, expression of DCC is lost during ovarian tumorigenesis (62). Our cultured luteal fibroblast-like cells and luteinized granulosa cells also express Netrin-1 and DCC (data not shown) so they have the capacity for this signaling pathway to be activated.

The binding and sequestering of SLIT by the ROBO1/Fc chimera would potentially prevent the ROBO/DCC interaction. Under these conditions Netrin-1 would be able to interact with DCC and block apoptosis. Interestingly, past research has suggested that hCG works as a prosurvival factor by regulating known apoptosis factors such as the Fas-Fas ligand system, Bcl-2 and Bax system, and survivin (2). Our results suggest the SLIT-ROBO system should also be added to this list. Therefore, the absence of hCG action could be a signal to initiate luteal cell apoptosis when pregnancy does not occur.

Our laboratory has previously shown that the glucocorticoid cortisol may have a role in maintaining CL structure and function during early pregnancy (49). We showed that hCG could stimulate the expression of 11βHSD type 1 and reduce 11βHSD type 2 expression, resulting in increased local cortisol generation. Interestingly, in these studies, cortisol increased the survival of luteinized granulosa cells treated with the cortisol and progesterone antagonist RU486. In our present study, we found that cortisol could significantly reduce SLIT2 and SLIT3 expression in short-term in vitro cultures of human luteinized granulosa cells and luteal fibroblast-like cells. Previous research has suggested that cortisol can protect cultured luteinized granulosa cells from bcl-2 and TNF-{alpha}-induced apoptosis (63). Perhaps, during early stages of pregnancy, increased cortisol production also protects luteal cells from SLIT/ROBO/DCC-mediated apoptosis. The molecular mechanism and relevance of the reduction in SLIT2 and SLIT3 expression by cortisol, however, requires further elucidation.

In conclusion, we have identified a system, the SLIT-ROBO pathway, that could play a novel role in human luteolysis. The mechanism by which the SLIT and ROBO proteins do this could involve promoting apoptosis and controlling migration of a number of cell types, including fibroblasts and luteinized granulosa cells. We also believe that these molecules are negatively regulated by hCG and possibly cortisol during luteal rescue. This may facilitate maintenance of the structure of the CL during early maternal recognition of pregnancy.


   Acknowledgments
 
The authors thank the patients, clinical fellows, embryologists, and nursing staff of the Edinburgh Assisted Conception Unit for help in sample collection. We are also grateful to Professor Hamish M. Fraser for support in human CL tissue collection and Professor Richard A. Anderson for human fetal ovary RNA samples. In addition we appreciate the technical advice and support of Dr. Sander van den Driesche, Dr. Brigid Orr, and Dr. Griet Vanpoucke. Furthermore, we thank Dr. Axel Thomson for very helpful intellectual discussion.


   Footnotes
 
R.E.D. is supported by a Medical Research Council Career Development Fellowship.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online June 19, 2008

Abbreviations: CL, Corpus luteum; DCC, deleted in colorectal cancer; FBS, fetal bovine serum; G6PDH, glucose-6-phosphate dehydrogenase; hCG, human chorionic gonadotropin; 11βHSD, 11β-hydroxysteroid dehydrogenase; LDL, low-density lipoprotein; LGC, luteinized granulosa cell; MMP, matrix metalloproteinase; NRS, normal rabbit serum; ROBO, roundabout.

Received February 12, 2008.

Accepted for publication June 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Behrman HR, Endo T, Aten RF, Musicki B 1993 Corpus luteum function and regression. Reprod Med Rev 2:153–180
  2. Fraser HM, Lunn SF, Harrison DJ, Kerr JB 1999 Luteal regression in the primate: different forms of cell death during natural and gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis. Biol Reprod 61:1468–1479[Abstract/Free Full Text]
  3. Duncan WC, Hillier SG, Gay E, Bell J, Fraser HM 2005 Connective tissue growth factor expression in the human corpus luteum: paracrine regulation by human chorionic gonadotropin. J Clin Endocrinol Metab 90:5366–5376[Abstract/Free Full Text]
  4. Duncan WC, McNeilly AS, Illingworth PJ 1998 The effect of luteal "rescue" on the expression and localization of matrix metalloproteinases and their tissue inhibitors in the human corpus luteum. J Clin Endocrinol Metab 83:2470–2478[Abstract/Free Full Text]
  5. Duncan WC, Rodger FE, Illingworth PJ 1998 The human corpus luteum: reduction in macrophages during simulated maternal recognition of pregnancy. Hum Reprod 13:2435–2442[Abstract/Free Full Text]
  6. Duncan WC 2000 The human corpus luteum: remodelling during luteolysis and maternal recognition of pregnancy. Rev Reprod 5:12–17[Abstract]
  7. Seeger M, Tear G, Ferres-Marco D, Goodman CS 1993 Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron 10:409–426[CrossRef][Medline]
  8. Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, Tessier-Lavigne M, Kidd T 1999 Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96:795–806[CrossRef][Medline]
  9. Li HS, Chen JH, Wu W, Fagaly T, Zhou L, Yuan W, Dupuis S, Jiang ZH, Nash W, Gick C, Ornitz DM, Wu JY, Rao Y 1999 Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 96:807–818[CrossRef][Medline]
  10. Huminiecki L, Gorn M, Suchting S, Poulsom R, Bicknell R 2002 Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis. Genomics 79:547–552[CrossRef][Medline]
  11. Itoh A, Miyabayashi T, Ohno M, Sakano S 1998 Cloning and expressions of three mammalian homologues of Drosophila slit suggest possible roles for Slit in the formation and maintenance of the nervous system. Brain Res 62:175–186[CrossRef]
  12. Dickson BJ 2002 Molecular mechanisms of axon guidance. Science 298:1959–1964[Abstract/Free Full Text]
  13. Piper M, Georgas K, Yamada T, Little M 2000 Expression of the vertebrate Slit gene family and their putative receptors, the Robo genes, in the developing murine kidney. Mech Dev 94:213–217[CrossRef][Medline]
  14. Clark K, Hammond E, Rabbitts P 2002 Temporal and spatial expression of two isoforms of the Dutt1/Robo1 gene in mouse development. FEBS Lett 523:12–16[CrossRef][Medline]
  15. Anselmo MA, Dalvin S, Prodhan P, Komatsuzaki K, Aidlen JT, Schnitzer JJ, Wu JY, Kinane TB 2003 Slit and robo: expression patterns in lung development. Gene Expr Patterns 3:13–19[CrossRef][Medline]
  16. Park KW, Morrison CM, Sorensen LK, Jones CA, Rao Y, Chien CB, Wu JY, Urness LD, Li DY 2003 Robo4 is a vascular-specific receptor that inhibits endothelial migration. Dev Biol 261:251–267[CrossRef][Medline]
  17. Suchting S, Heal P, Tahtis K, Stewart LM, Bicknell R 2005 Soluble Robo4 receptor inhibits in vivo angiogenesis and endothelial cell migration. FASEB J 19:121–123[Abstract/Free Full Text]
  18. Seth P, Lin Y, Hanai J, Shivalingappa V, Duyao MP, Sukhatme VP 2005 Magic roundabout, a tumor endothelial marker: expression and signaling. Biochem Biophys Res Commun 332:533–541[Medline]
  19. Xian J, Clark KJ, Fordham R, Pannell R, Rabbitts TH, Rabbitts PH 2001 Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene. Proc Natl Acad Sci USA 98:15062–15066[Abstract/Free Full Text]
  20. Yuan W, Rao Y, Babiuk RP, Greer JJ, Wu JY, Ornitz DM 2003 A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3. Proc Natl Acad Sci USA 100:5217–5222[Abstract/Free Full Text]
  21. Liu J, Zhang L, Wang D, Shen H, Jiang M, Mei P, Hayden PS, Sedor JR, Hu H 2003 Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice. Mech Dev 120:1059–1070[CrossRef][Medline]
  22. Grieshammer U, Le M, Plump AS, Wang F, Tessier-Lavigne M, Martin GR 2004 SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Developmental cell 6:709–717[CrossRef][Medline]
  23. Chen B, Blair DG, Plisov S, Vasiliev G, Perantoni AO, Chen Q, Athanasiou M, Wu JY, Oppenheim JJ, Yang D 2004 Cutting edge: bone morphogenetic protein antagonists Drm/Gremlin and Dan interact with Slits and act as negative regulators of monocyte chemotaxis. J Immunol 173:5914–5917[Abstract/Free Full Text]
  24. Wu JY, Feng L, Park HT, Havlioglu N, Wen L, Tang H, Bacon KB, Jiang Z, Zhang X, Rao Y 2001 The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410:948–952[CrossRef][Medline]
  25. Liu D, Hou J, Hu X, Wang X, Xiao Y, Mou Y, De Leon H 2006 Neuronal chemorepellent Slit2 inhibits vascular smooth muscle cell migration by suppressing small GTPase Rac1 activation. Circ Res 98:480–489[Abstract/Free Full Text]
  26. Kanellis J, Garcia GE, Li P, Parra G, Wilson CB, Rao Y, Han S, Smith CW, Johnson RJ, Wu JY, Feng L 2004 Modulation of inflammation by slit protein in vivo in experimental crescentic glomerulonephritis. Am J Pathol 165:341–352[Abstract/Free Full Text]
  27. Dallol A, Dickinson RE, Latif F 2005 Epigenetic disruption of the SLIT-ROBO interactions in human cancer. In: Esteller M, ed. DNA methylation, epigenetics and metastasis. Dorvecht, The Netherlands: Springer Netherlands; 191–214
  28. Chedotal A, Kerjan G, Moreau-Fauvarque C 2005 The brain within the tumor: new roles for axon guidance molecules in cancers. Cell Death Differ 12:1044–1056[CrossRef][Medline]
  29. Dallol A, Da Silva NF, Viacava P, Minna JD, Bieche I, Maher ER, Latif F 2002 SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res 62:5874–5880[Abstract/Free Full Text]
  30. Dallol A, Forgacs E, Martinez A, Sekido Y, Walker R, Kishida T, Rabbitts P, Maher ER, Minna JD, Latif F 2002 Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers. Oncogene 21:3020–3028[CrossRef][Medline]
  31. Mitra AB, Murty VV, Li RG, Pratap M, Luthra UK, Chaganti RS 1994 Allelotype analysis of cervical carcinoma. Cancer Res 54:4481–4487[Abstract/Free Full Text]
  32. Narayan G, Goparaju C, Arias-Pulido H, Kaufmann AM, Schneider A, Durst M, Mansukhani M, Pothuri B, Murty VV 2006 Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression. Mol Cancer 5:16[CrossRef][Medline]
  33. Pulido HA, Fakruddin MJ, Chatterjee A, Esplin ED, Beleno N, Martinez G, Posso H, Evans GA, Murty VV 2000 Identification of a 6-cM minimal deletion at 11q23.1–23.2 and exclusion of PPP2R1B gene as a deletion target in cervical cancer. Cancer Res 60:6677–6682[Abstract/Free Full Text]
  34. Singh RK, Indra D, Mitra S, Mondal RK, Basu PS, Roy A, Roychowdhury S, Panda CK 2007 Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene. Hum Genet 122:71–81[CrossRef][Medline]
  35. Dickinson RE, Dallol A, Bieche I, Krex D, Morton D, Maher ER, Latif F 2004 Epigenetic inactivation of SLIT3 and SLIT1 genes in human cancers. Br J Cancer 91:2071–2078[CrossRef][Medline]
  36. Faulkner SW, Friedlander ML 2000 Molecular genetic analysis of malignant ovarian germ cell tumors. Gynecol Oncol 77:283–288[CrossRef][Medline]
  37. Dallol A, Krex D, Hesson L, Eng C, Maher ER, Latif F 2003 Frequent epigenetic inactivation of the SLIT2 gene in gliomas. Oncogene 22:4611–4616[CrossRef][Medline]
  38. Dallol A, Morton D, Maher ER, Latif F 2003 SLIT2 axon guidance molecule is frequently inactivated in colorectal cancer and suppresses growth of colorectal carcinoma cells. Cancer Res 63:1054–1058[Abstract/Free Full Text]
  39. Morrissey C, Dallol A, Latif F, Gazdar A, Minna JD, The candidate tumour suppressor gene SLIT-2 suppresses growth and induces apoptosis in lung and breast cancer. Proc 95th Annual Meeting American Association for Cancer Research, Orlando, FL, 2004
  40. Prasad A, Fernandis AZ, Rao Y, Ganju RK 2004 Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells. J Biol Chem 279:9115–9124[Abstract/Free Full Text]
  41. Werbowetski-Ogilvie TE, Seyed Sadr M, Jabado N, Angers-Loustau A, Agar NY, Wu J, Bjerkvig R, Antel JP, Faury D, Rao Y, Del Maestro RF 2006 Inhibition of medulloblastoma cell invasion by Slit. Oncogene 25:5103–5112[Medline]
  42. Xian J, Aitchison A, Bobrow L, Corbett G, Pannell R, Rabbitts T, Rabbitts P 2004 Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter. Cancer Res 64:6432–6437[Abstract/Free Full Text]
  43. Bayne RA, Martins da Silva SJ, Anderson RA 2004 Increased expression of the FIGLA transcription factor is associated with primordial follicle formation in the human fetal ovary. Mol Hum Reprod 10:373–381[Abstract/Free Full Text]
  44. Duncan WC, Gay E, Maybin JA 2005 The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro. Reproduction 130:83–93[Abstract/Free Full Text]
  45. Ratcliffe KE, Anthony FW, Richardson MC, Stones RW 1999 Morphology and functional characteristics of human ovarian microvascular endothelium. Hum Reprod 14:1549–1554[Abstract/Free Full Text]
  46. Jacobs M, Levin M 2002 An improved endothelial barrier model to investigate dengue haemorrhagic fever. J Virol Methods 104:173–185[CrossRef][Medline]
  47. Matsubara H, Ikuta K, Ozaki Y, Suzuki Y, Suzuki N, Sato T, Suzumori K 2000 Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells. J Clin Endocrinol Metab 85:1620–1626[Abstract/Free Full Text]
  48. Roby KF, Weed J, Lyles R, Terranova PF 1990 Immunological evidence for a human ovarian tumor necrosis factor-{alpha}. J Clin Endocrinol Metab 71:1096–1102[Abstract/Free Full Text]
  49. Myers M, Lamont MC, van den Driesche S, Mary N, Thong KJ, Hillier SG, Duncan WC 2007 Role of luteal glucocorticoid metabolism during maternal recognition of pregnancy in women. Endocrinology 148:5769–5779[CrossRef][Medline]
  50. Myers M, Gay E, McNeilly AS, Fraser HM, Duncan WC 2007 In vitro evidence suggests activin-A may promote tissue remodeling associated with human luteolysis. Endocrinology 148:3730–3739[CrossRef][Medline]
  51. Suchting S, Bicknell R, Eichmann A 2006 Neuronal clues to vascular guidance. Exp Cell Res 312:668–675[CrossRef][Medline]
  52. Wong K, Park HT, Wu JY, Rao Y 2002 Slit proteins: molecular guidance cues for cells ranging from neurons to leukocytes. Curr Opin Genet Dev 12:583–591[CrossRef][Medline]
  53. Kaczmarek MM, Kowalczyk AE, Waclawik A, Schams D, Ziecik AJ 2007 Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy. Mol Reprod Dev 74:730–739[CrossRef][Medline]
  54. Xu J, Hennebold JD, Stouffer RL 2006 Dynamic expression and regulation of the corticotropin-releasing hormone/urocortin-receptor-binding protein system in the primate ovary during the menstrual cycle. J Clin Endocrinol Metab 91:1544–1553[Abstract/Free Full Text]
  55. Tanno T, Fujiwara A, Takenaka S, Kuwamura M, Tsuyama S 2005 Expression of a chemorepellent factor, Slit2, in peripheral nerve regeneration. Biosci Biotechnol Biochem 69:2431–2434[CrossRef][Medline]
  56. Bashaw GJ, Kidd T, Murray D, Pawson T, Goodman CS 2000 Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101:703–715[CrossRef][Medline]
  57. Wong K, Ren XR, Huang YZ, Xie Y, Liu G, Saito H, Tang H, Wen L, Brady-Kalnay SM, Mei L, Wu JY, Xiong WC, Rao Y 2001 Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107:209–221[CrossRef][Medline]
  58. Sugino N, Okuda K 2007 Species-related differences in the mechanism of apoptosis during structural luteolysis. J Reprod Dev 53:977–986[CrossRef][Medline]
  59. Khan SM, Dauffenbach LM, Yeh J 2000 Mitochondria and caspases in induced apoptosis in human luteinized granulosa cells. Biochem Biophys Res Commun 269:542–545[CrossRef][Medline]
  60. Stein E, Tessier-Lavigne M 2001 Hierarchical organization of guidance receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science 291:1928–1938[Abstract/Free Full Text]
  61. Forcet C, Ye X, Granger L, Corset V, Shin H, Bredesen DE, Mehlen P 2001 The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation. Proc Natl Acad Sci USA 98:3416–3421[Abstract/Free Full Text]
  62. Saegusa M, Machida D, Okayasu I 2000 Loss of DCC gene expression during ovarian tumorigenesis: relation to tumour differentiation and progression. Br J Cancer 82:571–578[CrossRef][Medline]
  63. Sasson R, Amsterdam A 2003 Pleiotropic anti-apoptotic activity of glucocorticoids in ovarian follicular cells. Biochem Pharmacol 66:1393–1401[CrossRef][Medline]



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R. E. Dickinson, A. J. Stewart, M. Myers, R. P. Millar, and W. C. Duncan
Differential Expression and Functional Characterization of Luteinizing Hormone Receptor Splice Variants in Human Luteal Cells: Implications for Luteolysis
Endocrinology, June 1, 2009; 150(6): 2873 - 2881.
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