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BOR - Papers in Press, published online ahead of print February 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036780
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BIOLOGY OF REPRODUCTION 73, 20–28 (2005)
DOI: 10.1095/biolreprod.104.036780
© 2005 by the Society for the Study of Reproduction, Inc.

Spatiotemporal Expression of Heparanase During Human and Rodent Ovarian Folliculogenesis1

Ronit Haimov-Kochman 3, Diana Prus 4, Eyal Zcharia 5, Debra S. Goldman-Wohl 3, Shira Natanson-Yaron 3, Caryn Greenfield 3, Eyal Y. Anteby 3, Reuven Reich 6, Joseph Orly 7, Alex Tsafriri 8, Arye Hurwitz 3, Israel Vlodavsky 2 9, and Simcha Yagel 3

Departments of Obstetrics and Gynecology,3 Pathology,4 Oncology,5 Hadassah University Medical Center, Jerusalem 91240, Israel Department of Clinical Pharmacology,6 The Hebrew University, Jerusalem 91120, Israel Department of Biological Chemistry,7 The Alexander Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel Department of Biological Regulation,8 Weizmann Institute of Science, Rehovot 76100, Israel The Cancer and Vascular Biology Research Center,9 The Bruce Rappaport Faculty of Medicine, Haifa 31096, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs), major components of the basement membrane (BM) and extracellular matrix (ECM). Heparanase activity results in release of HSPG-bound molecules, including basic fibroblast growth factor (FGF2). Structural and functional development of the corpus luteum (CL) involves tissue remodeling, active angiogenesis, and steroid production. Heparanase-induced ECM and BM breakdown as well as FGF2-stimulated endothelial proliferation may have an important role in the regulation of luteal function. Heparanase mRNA was detected by reverse-transcription-polymerase chain reaction in granulosa cells recovered from follicular fluid of in vitro fertilization patients. Using sulfate-labeled ECM, heparanase enzymatic activity was determined in human luteinized granulosa cells. Employing immunohistochemistry, heparanase protein was localized predominantly in the theca interna cell layer of the mature antral follicle, whereas in human corpora lutea, both luteinized granulosa and theca cells were immunostained for heparanase. During luteolysis, heparanase was identified in macrophages surrounding the forming corpus albicans. In serially sectioned ovaries from unstimulated rats as well as from eCG-treated rats, expression of heparanase was noted exclusively in the ovarian steroid-producing interstitial tissue. Following an ovulatory dose of hCG, heparanase was immunostained also in lutein cells of the forming corpora lutea. Temporal expression of heparanase in granulosa cells during the luteal phase and in macrophages during luteal regression supports the hypothesis that heparanase plays a role in human ovarian ECM remodeling and may potentiate cellular migration and growth factor bioavailability.

basic fibroblast growth factor, corpus luteum, follicle, granulosa cells, heparanase, ovary, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparanase

Interactions between the extracellular environment and cells influence cellular proliferation, differentiation, and migration. Heparan sulfate proteoglycans (HSPGs) are major components of the extracellular matrix (ECM) and the basement membranes (BM) and hence play an important role in these interactions. HSPGs bind structural proteins, such as collagen, laminin, and fibronectin, as well as various growth and differentiation promoting factors, including basic fibroblast growth factor (FGF2), vascular endothelial growth factor (VEGF), and platelet-derived growth factor [1–4]. Heparan sulfate (HS)-degrading endoglycosidase, commonly referred to as heparanase, cleaves the HS side chains of HSPGs [5–9]. Heparanase was shown to play a pivotal role in remodeling of the BM and ECM during injury, inflammation, and cancer metastasis and in regulating cell growth, differentiation, and angiogenesis by releasing growth factors that are bound to HSPGs [7–11]. Degradation of the BM in sites of tissue remodeling could allow extravasation of migrating fibroblasts and endothelial and tumor cells [7–11]. Localization of heparanase to endothelial cells of small vessels [11, 12] further supports the hypothesis of its role in angiogenesis. A direct correlation was found between expression of heparanase, the metastatic potential of cancer cells, and tumor vascularity [7–11].

Corpus Luteum

Transformation of a fluid-filled preovulatory follicle into a solid, progesterone-producing corpus luteum (CL) occurs after ovulation. The preovulatory follicle is compartmentalized into a highly vascular theca layer and a nonvascular granulosa layer separated by a BM and independently regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [13, 14]. Definitive structural and functional changes occur in these two compartments around the time of ovulation. Shortly before ovulation, the BM breaks down, and the capillaries at the inner capillary plexus of the theca interna rapidly sprout and invade the inner granulosa cell layer [13–15]. During ovulation, the follicular wall ruptures, and the follicle collapses to form folds that interdigitate with the luteinizing granulosa cells. The growth rate of the proliferating cells of the early developing CL is extremely rapid and comparable to that of the most aggressive tumors [13]. A complex capillary network is formed, bringing each granulosa-lutein cell into close approximation to the vascular bed. Approximately 50% of the cells of the mature CL are endothelial cells [14, 15]. The vascular development of the CL is regulated by angiogenic factors, including VEGF [14–18], angiopoetins (ANGP1, ANGP2) [14, 18–20], and FGF2 [21–25]. FGF2 mRNA synthesis is stimulated by LH in bovine CL and in cultured luteal cells [22]. During human CL formation, FGF2 is localized in granulosa, and theca lutein cells [23], FGF receptors (FGFR1 and 2), were demonstrated in the parenchyma as well as the vasculature of the CL [24]. During luteolysis, the regressing CL is characterized over a few days by a rapid phase of tissue disintegration and over several weeks by a slow phase of tissue remodeling, macrophage infiltration, and fibroblastic growth. Later, the CL completely disappears, leaving a small hyaline scar, the corpus albicans [15].

Hence, extensive remodeling of the ECM and loss of integrity of the BM occur during the development of the CL [26]. As a reservoir of a variety of biologically active factors, the ECM modulates cellular proliferation, differentiation, and migration [9, 13]. ECM-degrading proteinases, such as matrix metalloproteinases (MMPs) and plasminogen activators (PLAT and PLAU), were detected in luteizing theca and granulosa cells of corpora lutea [27, 28].

To date, in spite of the established role of heparanase in ECM remodeling, cell invasion, and angiogenesis, its involvement in normal follicular and luteal development is largely unknown. The present study was undertaken to analyze the localization and possible role of heparanase in the transformation of an ovarian follicle into a CL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Granulosa Cell Collection

Use of discarded material was approved by the Hadassah Medical Center review board. Granulosa cells were retrieved from large follicles obtained from normal ovulatory women undergoing ovulation induction for in vitro fertilization (IVF), as previously described [29]. Briefly, these women were pretreated with gonadotropin-releasing hormone analogue (Decapeptil; Ferring Pharmaceutical, Malmo, Sweden). Treatment started in the midluteal phase of the preceding cycle and continued for 14 days. Recombinant FSH (Serono S.A., Aubonne, Switzerland) was started thereafter, at a dose of 3 ampoules/day for 5 days and subsequently adjusted according to the individual response. The hCG (10 000 IU) was administered when at least two follicles were 18 mm in diameter. All accessible follicles were harvested by transvaginal ultrasonographic aspiration, 36 h following hCG administration, and oocytes were removed from the follicular aspirates [28]. The follicular aspirates were layered on 10 ml of histopac columns (Sigma-Aldrich Corporation, St. Louis, MO) and centrifuged at 700 x g for 30 min to remove the red blood cells. Thereafter, the cells were aspirated from the interface, gently pipetted, resuspended, and counted in a hemocytometer. Viability, tested by trypan blue, was >90%.

Human Granulosa Cell Culture

The culture procedure was previously described by Hurwitz et al. [29]. Briefly, granulosa cells obtained from women undergoing IVF (250 000 cells/well; NUNC Brand Products, Halge Nunc International, Denmark) were maintained for 48 h in RPMI medium (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 10% fetal calf serum and subsequently cultured (5% CO2, 37°C) for 48 h in serum-free media in humidified incubator.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

RNA was isolated with TRizol (Life Technologies, Grand Island, NY) according to the manufacturer's instructions and was quantitated by ultraviolet absorption. After reverse transcription of 500 ng total RNA by oligo (dT) priming, the resulting single-stranded cDNA was amplified using TaqDNA polymerase and buffer (Promega, Madison, WI) [5, 11]. Oligonucleotide primers HPU-355 (5'-TTCGATCCCAAGAAGGAATCAAC-3') and HPL-229 (5'-CTAGTGATGCCATGTAACTGAATC-3') were used to amplify HPSE by RT-PCR [5, 12]. The PCR conditions were initial denaturation of 4 min at 94°C and subsequent denaturation for 45 sec at 94°C, annealing for 1 min at 60°C and extension for 1 min at 72°C (26 cycles) [5]. Aliquots of 10 µl of the amplification products were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.

Heparanase Activity Assay

Preparation of Na2[35S]O4-labeled ECM-coated dishes and determination of heparanase activity were performed as described in detail elsewhere [5, 30, 31]. Briefly, [35S]O4 (540–590 mCi/mmol) was added (25 µCi/ml) to cultured bovine corneal endothelial cells, 2 and 5 days after seeding, and the cultures were incubated with the label without medium change. Between 6 and 8 days after the cells reached confluence, the subendothelial ECM was exposed by lysing (3 min, 22°C) the cell layer with a solution containing 0.5% Triton-X100 and 20 mM NH4OH in phosphate-buffered saline (PBS). The ECM remained intact, free of cellular debris, and firmly attached to the entire area of the tissue culture dish. Granulosa cells (1 x 106 cells/ml) were subjected to three cycles of freezing and thawing in heparanase reaction mixture (50 mM NaCl, 1 mM CaCl2, 1 mM DTT, 20 mM buffer phosphate citrate, pH 6.0). Cell lysates or follicular fluid (1 ml) were incubated (24 h, 37°C, pH 6.2) with 35S-labeled ECM. The incubation medium containing labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS and their radioactivity counted in a ß-scintillation counter. Degradation fragments of HS side chains are eluted at 0.5 < Kav < 0.8 (fractions 12–30). Nearly intact HSPGs are eluted just after the Vo (Kav < 0.2, fractions 2–10). Each experiment was performed three times, and the variation in elution positions (Kav values) did not exceed ±15% [5, 30, 31].

Human Tissue Preparation

The study was approved by the Hadassah Medical Center Institutional Human Research Committee. Tissue specimens from 10 premenopausal, regularly cycling nonpregnant subjects aged 16–52 yr old, undergoing either elective or urgent operations, were retrospectively collected. The patients had no known previous ovarian pathologies or history of infertility. They were admitted for either cystectomy or oophorectomy for various diagnoses, such as hemorrhagic CL cyst or complicated ovarian cystic mass or as an accompanying procedure to total abdominal hysterectomy for a variety of reasons excluding malignancies. Ten to 15 tissue specimens from each subject were formalin fixed and paraffin embedded. Sections (4 µm) were either stained with hematoxylin-eosin or prepared for immunohistochemistry studies.

Staging of Human Ovaries

Ovarian follicles stained with hematoxylin-eosin were staged by a pathologist with an expertise in gynecologic pathology and classified according to basic morphological criteria. The ovaries were examined in terms of the following parameters: size of the follicles, ovulation, or subsequent hemorrhage; size, color, and tissue consistency of the developing CL; and signs of CL regression, including the appearance of fibroblasts in the residual CL. According to these histological criteria, follicles were grouped into the following categories: primary follicle, preantral follicle, antral follicle, mature antral follicle, early CL, mature midstage CL, regressing CL, and corpus albicans [32]. Maturity of antral follicles was determined on the assumption of an eccentric position of the oocyte at one pole of the follicle and the formation of the cumulus oophorous by granulosa cells encircling the oocyte and protruding into the antrum. Atretic follicles were also identified. Subsequently, a stage-defined archive of cyclic follicles was established. Given the well-characterized histology of the ovarian cycle, follicles could reliably be staged within 2–3 days by morphologic criteria [32].

Animals

Intact, immature female WISTAR rats were obtained from Harlan Laboratories (Jerusalem, Israel) and maintained in 16L:8D schedule with food and water ad libitum. Animals were treated in accordance with the National Research Council (NRC) publication Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science). All protocols had the approval of the Institutional Committee on Animal Care and Use, School of Medicine, The Hebrew University of Jerusalem.

Hormone-Induced Ovulating Rats

Prepubertal 24–27-day-old WISTAR rat females were hyperstimulated by intraperitoneal injection of 5 IU equine chorionic gonadotropin (eCG) at noon, and 4 IU hCG were administered 48 h later. The animals were killed by cervical dislocation at specific times—0 h, 24 h post-eCG, 48 h post-eCG, 12 h post-hCG, 24 h post-hCG, and 48 h post-hCG—and their ovaries were removed for further evaluation.

Immunohistochemistry

Sections (4 µm) of retrospectively collected formalin-fixed paraffin-embedded human ovarian tissue and formalin-fixed paraffin-embedded rat ovaries were analyzed by immunostaining using a polyclonal rabbit anti-human heparanase antibody (Ab-p3) that is cross-reactive with both the human and the rat antigens [6]. These antibodies were raised against a peptide (R273KTAKMLKSFLKAGGEVI290) located in the 50-kDa subunit of heparanase and were kindly provided by Dr. Robert L. Heinrikson (Pfizer, Kalamazoo, MI) [33]. This peptide was conjugated to keyhole limpet hemocyanin (KLH) and injected into rabbits. Specificity of this antibody was verified by immunoblot analysis of crude platelet preparation [33]. We have also used polyclonal rabbit anti-heparanase antibodies (733) directed against a synthetic peptide (158KKFKNSTYRSSSVD171) corresponding to the N-terminus of the 50-kDa subunit of the heparanase enzyme. Similar immunostaining pattern was obtained with the two antibodies [34]. The sections of human ovarian tissue were further subjected to immunostaining employing 3G10 anti-HS antibodies (kindly provided by the late Dr. M. Bernfield, Division of Newborn Medicine, Children Hospital, Boston, MA) [35] and monoclonal anti-human FGF2 antibodies (R&D Systems).

The localization of heparanase, HS, and FGF2 was evaluated in the different cellular subpopulations. These subpopulations were further characterized by employing antibodies directed against steroidogenic acute regulatory protein (STAR) (kindly provided by Dr. Douglas Stocco, Texas Tech University Health Sciences Center, Lubbock, TX) for steroid-producing cells, CD34 (Clone QBEnd 10, DAKO, Carpinteria, CA) for human endothelial cells, and CD68 (Clone PG-M1, DAKO) for human macrophages. Immunohistochemical staining for STAR was performed using polyclonal rabbit antiserum raised against a peptide fragment (amino acids 88–98) of the 30-kDa mouse STAR protein [36]. Anti-STAR serum is cross-reactive with human and rat antigens. STAR is a specific marker for steroidogenesis, as it facilitates transfer of cholesterol substrate into the inner mitochondrial membranes, where progesterone biosynthesis takes place.

Immunohistochemistry for Heparanase

Immunohistochemistry for heparanase was performed as previously described [12, 37]. Briefly, 4-µm sections were deparaffinized and rehydrated. Tissue was then denatured for 3 min in a microwave oven in citrate buffer (0.01 M, pH 6.0). The slides were washed, blocked, and incubated at room temperature with the primary antibody using the HISTOSTAIN-PLUS kit (Zymed Laboratories Inc, San Francisco, CA). Sections were incubated with the previously mentioned polyclonal rabbit anti-human heparanase antibody (Ab-p3) diluted 1:150 or with DMEM medium supplemented with 10% horse serum as control, followed by incubation (30 min) with EnVision, peroxidase-conjugated anti-rabbit antibodies (DAKO). Color was developed using substrate-chromagen solution of aminoethyl carbazole-AEC (Sigma), followed by counterstain with Mayer hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Heparanase in Follicular Lutein Granulosa Cells

RT-PCR was applied to evaluate expression of the HPSE gene by granulosa cells recovered from women undergoing IVF during oocyte retrieval 36 h after hCG exposure in vivo. For this purpose, total RNA was reverse transcribed and amplified using appropriate human heparanase primers. The expected 595-bp cDNA product of the human HPSE gene was clearly demonstrated in luteinizing granulosa cells (Gc) comparable to human placenta (P), previously shown to express high levels of heparanase [12] (Fig. 1, inset).



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  		FIG. 1. Heparanase activity in human granulosa cells lysate and in follicular fluid. Heparanase activity measured in human granulosa cell lysates (black squares) and in follicular fluid (white squares) originating from hCG-pre-exposed women undergoing in vitro fertilization. Granulosa cells were recovered during oocyte retrieval 36 h after hCG exposure in vivo. The follicular aspirates were collected and the granulosa cells centrifugated and lysed by three cycles of freezing and thawing. Granulosa cell lysates (black squares) and follicular fluid (white squares) were incubated (24 h, pH 6.2, 37°C) in serum-free medium in contact with sulfate-labeled ECM. The released labeled degradation fragments were analyzed by gel filtration on Sepharose 6B. Degradation fragments of heparan sulfate (HS) side chains are eluted at fractions 12–30 (0.5 < Kav < 0.8) as described in Materials and Methods. A similar elution profile was obtained when the incubation of the samples with ECM was performed in the absence or presence of protease inhibitors. Inset: RT-PCR. Total RNA isolated from hCG-preexposed granulosa cells (Gc) or from human placenta (P) was analyzed for expression of HPSE mRNA, using appropriate primers. The expected 595 bp cDNA product of the HPSE gene was demonstrated in luteinizing glanulosa cells (lane Gc) and human placenta (lane P). Lane M: 100-bp DNA molecular weight markers

Both luteinized granulosa cell lysates and follicular fluid recovered from oocyte retrieval 36 h following hCG administration were examined for heparanase activity. As demonstrated in Figure 1, heparanase activity was observed in both the granulosa cells and follicular fluid compartments as revealed by the release of HS degradation fragments from metabolically sulfate labeled ECM [30, 31]. In a control of buffer alone, there was no release of sulfate-labeled material eluted in fractions 12–30. We have previously demonstrated that labeled fragments eluted in fractions 12–30 are degradation products of HS, as they were 1) 5–6-fold smaller than intact HS side chains, 2) resistant to further digestion with papain and chondroitinase ABC, and 3) susceptible to deamination by nitrous acid. In contrast, sulfate-labeled material released from ECM by proteolytic enzymes is of much higher molecular weight, eluted in fractions 2–10 [30].

Localization of Heparanase in the Developing Human Ovarian Follicle and in the Corpus Luteum

The localization of heparanase in the cellular subpopulations of the developing ovarian follicle is summarized in Figures 2– 5. In primary, preantral and early antral follicles, heparanase protein was detected in the theca externa stromal cells surrounding the follicles (data not shown). Next, we investigated the localization of heparanase in mature antral follicles and in developing corpora lutea. Given the well-characterized histology of the ovarian cycle, a stage-defined hematoxylin-eosin-stained archive of cyclic follicles was established (Figs. 2A and 5A). In the mature follicle, heparanase was expressed in cells neighboring the BM, predominantly within the theca interna cells and to a lesser degree in the basal layers of the granulosa cells (Fig. 2, B and B'). HS, the heparanase substrate, was abundant at the BM separating the granulosa and the theca cell layers, on theca interna cells, and at the subendothelial BM of blood vessels (Fig. 2, D and D'). Thus, our data clarify that HS exists in target cells and BM of the mature antral follicle. Postovulatory lutein granulosa cells were immunostained for heparanase (Fig. 3) in both early (Fig. 3, top) and late (Fig. 3, bottom) stage CL. Both large lutein cells (granulosa cells in origin) and small lutein cells (theca interna cells in origin) express the heparanase protein (Fig. 3) from early stage CL to its regression (Fig. 4). Cytoplasmic granular staining of heparanase in lutein cells undergoing nuclear piknotic changes can be seen during involution of the CL (Fig. 4, B and B'). At the corpus albicans (Fig. 5), heparanase could still be identified in a granular pattern throughout the cell cytoplasm of CD68-positive macrophages scattered around the hyaline scar (Fig. 5C). In atretic follicles (Fig. 6), heparanase was immunodetected in nests of macrophages at close vicinity to the corpus fibrosum. A similar staining pattern was obtained using anti-heparanase antibodies Ab-p3 [33] (Figs. 2–6) and Ab #733 (not shown) directed against the N-terminus of the 50-kDa heparanase subunit and characterized by Zester et al., [34]. The identical staining pattern further supports the specificity of the heparanase immunostaining.



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  		FIG. 2. Immunodetection of heparanase and heparan sulfate (HS) in a mature antral follicle. A) Paraffin-embedded sections of human ovarian tissue were stained with hematoxylin-eosin, and a mature antral follicle was identified based on histological parameters, such as formation of cumulus oophorous, and assumption of an eccentric position of the ovum. Gc, Granulosa cells; Tc, Theca cells; Ov, ovum; B) Immunostaining of a section from the same series with anti-heparanase antibody. B') Higher magnification of the boxed area demonstrating heparanase expression in cells neighboring the basement membrane (BM), predominantly within the theca interna cells and to a lesser degree in the basal layers of the granulosa cells. C) Negative control of a section from the same series using only 10% horse serum without primary antibody. D and D') Consecutive section of mature antral follicle wall was immunostained with anti-heparan sulfate as formerly described [34]. HS is localized to the BM separating the granulosa and theca cell layers (arrows), the theca interna cells, and the subendothelial BM (asterisks). Original magnification A–C x100; B', D, and D' x400; and bar = 100 µm



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  		FIG. 5. Immunodetection of heparanase in the corpus albicans. A) Paraffin-embedded sections of human ovarian tissue were stained with hematoxylin-eosin. Corpus albicans was identified based on histological parameters, such as presence of central hyaline Scar (HySc) and the surrounding macrophages (Mac). B) Immunostaining of the consecutive section with anti-heparanase antibody showing that the macrophages are heparanase positive. B') In higher magnification of the boxed area, the enzyme is seen in a granular pattern throughout the cell cytoplasm. C) Immunostaining of the consecutive section with CD68, a specific marker for human macrophages. Original magnification A–C x25; B' x400; and bar = 100 µm



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  		FIG. 3. Immunodetection of heparanase in early and mature corpora lutea. A and C) Paraffin-embedded sections of human ovarian tissue were stained with hematoxylin-eosin. Corpora lutea were identified based on histological parameters, and the corpus luteum (CL) stage was defined according to presence of theca cell layer lobulation and of ovulatory cavity fibroblasts. LLGc, Large lutein granulosa cells; SLTc, Small lutein theca cells; OC, Ovulatory cavity. Top panel: early CL; bottom panel: late CL (magnification x100). B and D) Immunostaining of consecutive sections with anti-heparanase antibody. B' and D') Higher magnification of the boxed area in B and D, respectively, showing that in both early (B) and late stage (D) CL, the large lutein granulosa cells and small theca lutein cells strongly express heparanase. E) Negative control of a consecutive section using 10% horse serum without primary antibody. Original magnification A–D, x100; B' and D' x400; and bar = 100 µm



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  		FIG. 4. Immunodetection of heparanase in regressing corpus luteum (CL). A) Paraffin-embedded sections of human ovarian tissue were stained with hematoxylin-eosin. Regressing CL was identified based on histological parameters, such as presence of CL condensation and nuclear piknosis and abundant cytoplasmic lipid in lutein cells. LLGc, Large lutein granulosa cells; SLTc, Small lutein theca cells. B) Immunostaining of the consecutive section with anti-heparanase antibody showing that heparanase positive staining persisted within lutein granulosa and theca cells during the regression of the CL. B') In higher magnification of the boxed area, the enzyme is localized in a granular pattern throughout the cell cytoplasm. Arrows point to granulosa lutein cells undergoing pyknotic changes. C) Negative control of a consecutive section using 10% horse serum without primary antibody. Original magnification A–C x25; B' x400; and bar = 100 µm



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  		FIG. 6. Immunodetection of heparanase in the corpus fibrosum. A) Immunohistochemistry staining of paraffin-embedded sections of human ovarian tissue with anti-heparanase antibody showing an atretic follicle surrounded by macrophages (Mac). A') In higher magnification of the boxed area, the enzyme is in a granular cytoplasmic pattern localized in nests of macrophages in close vicinity to the corpus fibrosum. B) Immunostaining of the consecutive section with CD68, a specific marker for human macrophages. C) Negative control of a consecutive section using 10% horse serum without primary antibody. Original magnification A and C x25; B x100; A' x400; and bar = 100 µm

Together, these results indicate a spatiotemporal expression of heparanase by granulosa cells post luteinization and during regression of the CL and by macrophages during scarification and remodeling of the ovarian tissue.

Immunostaining of the CL with anti-heparanase (Fig. 7A) and anti-STAR (Fig. 7B) revealed that heparanase and STAR were localized to the same compartment of large and small lutein cells, indicating that heparanase is expressed in steroid-producing cells in the CL wall (Fig. 7). CD34-positive endothelial cells of developing small vessels entering the granulosa cell layer from the theca interna vascular wreath were heparanase negative (Fig. 8) and FGF2 positive (Fig. 9). FGF2 was also localized within cells of the theca interna and lutein granulosa cells of the CL; however, its expression was most prominent at the sprouting blood vessels (Fig. 9).



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  		FIG. 7. Localization of heparanase and steroidogenic acute regulatory protein (STAR) in the human corpus luteum (CL). Immunolocalization of (A) heparanase and (B) STAR (a marker of steroidogenic cells) in sections of human ovarian early CL wall. OC, Ovulatory cavity; OvS: Ovarian stroma. A' and B') Higher magnification of the boxed area in A and B, respectively, showing heparanase (A') and STAR (B') staining in lutein granulosa (LGc) and lutein theca cells (LTc), indicating that heparanase is expressed in steroid-producing cells. Original magnification A and B x100; A' and B' x400; and bar = 100 µm



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  		FIG. 8. Lack of expression of heparanase in sprouting capillaries of the corpus luteum (CL). A) Immunolocalization of heparanase in the developing human CL. Heparanase is preferentially expressed in lutein granulosa (LGc) and lutein theca cells (LTc) adjacent to proliferating blood vessels. OC, Ovulatory cavity; OvS, Ovarian stroma. A') Higher magnification of the boxed area demonstrating that small blood vessels (BV) within the theca interna layer (LTc) and throughout the luteinizing granulosa cell layer were heparanase negative. Original magnification A x100; A' x400; and bar = 100 µm



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  		FIG. 9. Immunolocalization of FGF2 in the human early corpus luteum (CL). A) Immunolocalization of FGF2 in human CL. The parenchyma of the CL consisting of theca and granulosa cells was mildly stained, while the endothelial cells of developing small vessels of the CL were strongly stained with anti-FGF2 antibodies. B) Negative control of consecutive section without primary antibody. A' and A'') Higher magnification of the boxed areas demonstrating FGF2-positive small blood vessels (arrows), identified by CD34, an endothelial cell specific marker (C). Original magnification A and B x25; A', A'', and C x100; and bar = 100 µm

Localization of Heparanase in Rodent Ovarian Folliculogenesis

To validate heparanase localization throughout the ovulatory cycle, we also examined the time-dependent expression of heparanase and STAR proteins in eCG/hCG-treated immature rats. In serially sectioned ovaries from unstimulated rats, as well as from eCG treated rats, expression of both heparanase and STAR was noted exclusively in the ovarian secondary interstitial tissue (Fig. 10, A–C, and A'– C'). Following an ovulatory dose of hCG, heparanase and STAR were expressed in lutein cells of the forming corpora lutea as well as in the steriodogenic interstitial tissue (Fig. 10, D–F, and D'–F'). Both proteins were also observed in the theca layers of follicles, which did not rupture. However, granulosa cells of the unruptured follicles did not express the two proteins (Fig. 10, E and E').



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  		FIG. 10. Immunolocalization of heparanase and STAR during rat follicular development and corpus luteum (CL) formation. Immunolocalization of heparanase (A–F) and STAR (A'–F') in serially sectioned ovaries from noninduced rats (A and A'), from animals sacrificed 24 and 48 h after eCG treatment (B, C, B', and C') or 12, 24, and 48 h following treatment with an ovulatory dose of hCG (D–F and D'–F'). In noninduced as well as eCG-treated rats, heparanase and STAR were noted exclusively in the ovarian interfollicular stroma and in the theca interna cells (Tc) (A–C and A'–C'). Following HCG administration, both proteins were strongly immunostained in lutein cells of the forming CL as well as in the steriodogenic interstitial tissue (D–F and D'–F'). Heparanase and STAR were also observed in the theca layers of follicles that did not rupture (asterisks). However, granulosa cells of unruptured follicles expressed none or only low levels of the two proteins (E and E'). Original magnification x100 and bar = 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CL development depends on dramatic tissue remodeling as well as sequential regulation of vascular outgrowth and regression. This process includes rapid proliferation of luteinized theca and granulosa cells along with a wave of vascular sprouting that eventually regress as the CL ages during luteolysis [13–15]. The extensive follicular tissue growth and vascularization is regulated in part by various HS-bound proliferation and angiogenesis promoting factors, such as FGF2 [14–25].

Here we report, for the first time, the presence of heparanase in normal human ovary. Heparanase mRNA, protein, and activity were demonstrated in human luteinized granulosa cells originating from follicular fluid of preovulatory follicles following hCG exposure. Immunostaining of human ovarian specimens clearly revealed that heparanase is expressed in the theca and granulosa cells of mature follicles. Moreover, HS, the sole substrate of heparanase, was found on theca interna cells and BM, suggesting the participation of heparanase in breakdown of the BM separating granulosa and theca cell layers and possibly in follicular rupture during ovulation. Indeed, increased gelatinolytic activity (MMPs 2 and 9) and marked up-regulation of serine proteases (PLAT and PLAU) were demonstrated following luteinization in both granulosa and theca cells [27, 28], testifying to the rapid turnover of BM components at this stage.

Heparanase catalytic activity was detected in the follicular fluid. This result could indicate that the enzyme is released into the follicular lumen by the granulosa cells, possibly enhancing follicular rupture. It appears that most of the enzyme remains intracellularly and may rather function in a polar manner, locally degrading the BM in response to the appropriate stimulus (i.e., LH). In fact, most cultured cells that degrade the ECM secrete little or no heparanase into the incubation medium. Platelets and cells of the immune system were shown to secrete heparanase primarily on activation [38]. A recent report localizes human heparanase to late endosomes and lysosomes [39], although the regulation of its secretion has not been elucidated yet.

The localization of heparanase in cell subpopulations throughout the ovarian cycle was studied in human and confirmed in the rodent ovulatory model. The detection of heparanase in the interstitial tissues and the theca layer of preantral, early antral, and mature antral follicles suggests that the enzyme may participate in early follicular progression through recruitment of blood vessels by facilitating the bioavailability of angiogenic factors sequestered in the ECM and BM. In the rat model, only granulosa cells of ruptured follicles expressed heparanase, whereas granulosa cells of unruptured follicles did not. The differential heparanase expression suggests that actual follicular rupture provokes tissue remodeling, where heparanase may play an active part. Concomitant with further follicular development into the CL, actively proliferating luteinized steroid-producing granulosa and theca cells express heparanase. Lutein cell proliferation in this stage may be attributed in part to heparanase-mediated release of ECM-resident active FGF2 and possibly other heparin-binding growth factors. FGF2 is a known mitogenic and survival factor to granulosa cells [21, 25]. Hence, the abundance of FGF2-positive small capillaries in the CL, in a close proximity to heparanase-producing luteinized cells, observed in our study suggests a cross talk between heparanase-producing lutein cells and FGF2-regulated endothelial cell proliferation. It is conceivable that LH induces heparanase production in lutein cells, which then stimulates their own growth in an autocrine manner and potentiates CL angiogenesis by paracrine mechanisms involving augmentation of growth factors bioavailability.

Lack of heparanase in endothelial cells of small blood vessels in the human and rodent CL is in contrast to previous findings of heparanase-positive endothelial cells of developing blood vessels in the villous placenta [12], in the chicken embryo [40], and in malignant tumors [11, 37]. This discordance could imply different mechanisms of angiogenesis in physiological (i.e., CL) versus fetal and malignant vascular development.

STAR was initially used in the rat model as a marker of steroidogenic cells with a known pattern of expression during folliculogenesis [41]. Surprisingly, heparanase and STAR were tightly coexpressed in various stages of follicular development in both rat and human. In the human ovary, both proteins were localized in the theca interna and granulosa cells of the CL. In rats, persistent expression of heparanase and STAR at the same cell compartment was noted all along the ovulatory cycle. Heparanase was expressed by the steroidogenic interstitial network and the theca interna cells in noninduced as well as in eCG-treated animals. Followed hCG administration, the expression of heparanase in the theca interna and granulosa cells paralleled steroid biosynthesis as was evidenced by STAR expression and was maximally pronounced 48 h following hCG exposure at the solid CL stage (Fig. 10). Moreover, STAR-negative granulosa cells of nonruptured follicles did not express heparanase. Similar spatial and temporal expression of heparanase and STAR during rodent follicular development following controlled ovarian hyperstimulation suggests that heparanase might be coregulated by steroid production pathways induced by FSH and LH.

Heparanase expression in luteinized granulosa and theca cells during CL regression may imply its active role in apoptosis. The association of heparanase and apoptosis was first suggested when heparanase expression in various ovarian malignancies was explored [42]. Markedly high concentrations of heparanase were noted in apoptotic cells of ovarian mucinous and endometriod carcinomas [41]. Recently, heparanase gene expression was reported to be correlated with spontaneous apoptosis in hepatocytes of cirrhotic liver and hepatocellular carcinoma [43]. Taken together, heparanase may contribute to the process of programmed cell death in the normal ovarian cycle.

The transformation of the CL into a hyaline scar of the corpus albicans coincides with atresia of small and large lutein cells, infiltration of macrophages, ingrowth of fibroblasts, and tissue reorganization [32]. Furthermore, heparanase-positive macrophages were also identified during follicular obliterative atresia and corpus fibrosum formation, occurring in large vesicular follicles that failed to ovulate. Heparanase expression in macrophages during ovarian scarification processes such as corpus albicans and corpus fibrosum formation may suggest its active role in ovarian tissue remodeling. Involvement of heparanase in tissue reorganization and morphogenesis was recently documented during wound repair and in the mammary glands of transgenic mice overexpressing the HPSE gene [44].

Heparanase localization in proliferating theca and granulosa cells during the establishment of the CL as well as in lutein cells and macrophages during luteolysis and atresia suggests a dual role of the enzyme in cell growth and apoptosis. Further investigation is under way to determine the role of heparanase in ovarian tissue remodeling during folliculogenesis and CL formation and regression.


   FOOTNOTES
 
1 Supported by grants from the Hadassah Medical Organization for research of women's health, from the Israeli Ministry of Health, and from the Salzberg Research Foundation. Back

2 Correspondence: Israel Vlodavsky, Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, P.O. Box 9649, Haifa 31096, Israel. FAX: 972 4 8523947; Vlodavsk{at}cc.huji.ac.il Back

Received: 2 October 2004.

First decision: 18 November 2004.

Accepted: 9 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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