A novel mechanism of vascular endothelial growth factor, leptin and ...

Human Reproduction vol.12 no.10 pp.2226–2234, 1997

A novel mechanism of vascular endothelial growth factor, leptin and transforming growth factor-β2 sequestration in a subpopulation of human ovarian follicle cells Michael Antczak, Jonathan Van Blerkom1 and Andrew Clark Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA 1To

whom correspondence should be addressed

This study describes the occurrence of a highly specialized subpopulation of granulosa and cumulus oophorus cells that accumulate and sequester specific growth factors by a novel mechanism. These cells are characterized by multiple balloon-like processes tethered to the cell by means of a slender stalk of plasma membrane. Time-lapse analyses demonstrate that these tethered structures (TS) form in minutes and frequently detach from the cell with the bulbous portion remaining motile on the cell surface. Serial section reconstruction of transmission electron microscopic images shows a specific and stable intracellular organization in which an apparent secretory compartment composed of densely packed vacuoles, vesicles, and cisternae is separated by a thick filamentous network from a nuclear compartment containing mitochondria, polyribosomes, lipid inclusions, and rough-surfaced endoplasmic reticulum. Immunofluorescent analysis performed during the formation of these structures showed a progressive accumulation of vascular endothelial growth factor, leptin, and transforming growth factor-β2 in the bulbous region. TS were identified in newly aspirated masses of granulosa and cumulus oophorus, and their production persists for months in culture. Observations of TS-forming cells made over several days of culture indicates that their production is episodic and factor release from these cells may be pulsatile. The findings suggest that a novel method of growth factor storage and release by an apparent apocrine-like mechanism occurs in the human ovarian follicle. The results are discussed with respect to possible roles in pre- and postovulatory follicular development. Key words: follicle cells/growth factor production and secretion/leptin/TGFβ2/VEGF

Introduction The biology of the ovarian follicle is a study of extremes. Largely inaccessible to general body circulation during preantral stages, it is quiescent for extended periods of time, yet it is capable of undergoing rapid cell expansion and changes in structure that reflect differentiation at the molecular and cellular levels in response to gonadotrophins. Each follicle that develops in the stimulated human ovary expresses a unique 2226

profile characterized by qualitative or quantitative differences in steroid hormone (Hartshorne, 1989), protein (Nayudu et al., 1989), dissolved oxygen content (Van Blerkom et al., 1997), and viable granulosa cell populations (Seifer et al., 1996). These variables may all be of developmental significance for the enclosed oocyte. The granulosa and cumulus oophorus of the growing follicle have a central role in establishing the intrafollicular environment prior to ovulation and in the formation of a functional corpus luteum after ovulation. The traditional view of granulosa cell function has changed considerably as the biology of these ovarian elements and the fluid to which they contribute have been subjected to more detailed analysis. In addition to a steroidogenic capacity, granulosa cells, including those of the cumulus oophorus, secrete a wide variety of growth factors [including basic fibroblast growth factor (bFGF), insulin-like growth factor-I and -II, epidermal growth factor (EGF), leukaemia inhibitory factor (LIF), transforming growth factor (TGF)-α and -β, tumour necrosis factor (TNF)-α: Echternkamp et al., 1990; Rabinovici et al., 1990; Zolti et al., 1990; Roberts and Skinner, 1991; Artini et al., 1994; Brannstrom et al., 1994; Li et al., 1994; Mason et al., 1994; Singh et al., 1995] and the cytokines interleukin (IL)-1 and IL-6 (Zolti et al., 1991; Ziltener et al., 1993). The expression of many of these macromolecules changes quantitatively or qualitatively, or both, during the preand post-ovulatory stages. Recently, two growth factors present at relatively high concentration in human follicular fluid have been identified as secretory products of granulosa and cumulus cells: Vascular endothelial growth factor (VEGF), which promotes endothelial cell expansion and vessel formation (Kamat et al., 1995; Van Blerkom et al., 1997) and leptin, a protein involved in very different regulatory activities including haematopoiesis (Bennett et al., 1996; Cioffi et al., 1996), fat deposition and metabolism (Zhang et al., 1994; Considine et al., 1996), and reproduction (Chehab et al., 1996; 1997; Cioffi et al., 1997). Here, we describe subpopulations of cells in both granulosa and cumulus oophorus which exhibit a remarkably active and unusual morphodynamic behaviour associated with the accumulation of VEGF, leptin, and TGFβ2. These cells form multiple balloon-like structures tethered to their apical surfaces by means of a slender stalk of plasma membrane. This unusual apocrine-like phenomenon was observed in newly recovered masses of human cumulus and granulosa cells and persisted in culture. We propose that tethered structure (TS) formation represents a novel mechanism of growth factor accumulation and sequestration which occurs in pre- and post-ovulatory human follicles, and we suggest this mechanism supports a unique mode of directed secretion which may play a role in supporting rapid bursts of cellular © European Society for Human Reproduction and Embryology

Growth factor accumulation and sequestration by ovarian follicle cells

expansion and differentiation during follicular growth and corpus luteum formation.

Materials and methods Cumulus and granulosa cell collection, culture and microscopic analysis Sheets of granulosa cells were retrieved from follicular fluid aspirates of women undergoing in-vitro fertilization (IVF) after controlled ovarian stimulation and ovulation induction (Van Blerkom et al., 1995), and cultured as previously described (Van Blerkom et al., 1997; Cioffi et al., 1997). Briefly, small sheets of granulosa cells free of any apparent thecal vessels were collected from each follicle aspirate with a micropipette, washed several times in culture medium, and dispersed by repeated passage through a 1 ml syringe fitted with a 22 gauge needle. Cell suspensions were plated onto uncoated clean and sterile glass coverslips or into plastic tissue culture vessels. The first light microscopic examination of these cultures was performed at 14 h. Cumulus oophorus fragments were removed from follicular fluid with a micropipette or obtained in small masses from the oocyte– cumulus complex by microneedle dissection. Some of the smaller fragments were examined immediately and continuously for at least 45 min by high resolution light microscopy to detect ‘wormlike’ movements along the surface indicative of TS. According to protocol, oocytes were inseminated in individual dishes with the first inspection for pronuclei performed 8–10 h later. After removal of the fertilized egg, each dish was examined under phase contrast optics, the adherent cumulus cells were washed twice with medium, then returned to culture. For some studies, oocyte-derived cumulus cells from multiple follicles were pooled prior to the establishment of long-term cultures. Cumulus and granulosa cell culture conditions and medium have been described in detail previously (Van Blerkom et al., 1997). Follicle cell cultures derived from 136 IVF patients were established for this study. Small fragments of cumulus oophorus or dispersed granulosa were placed in 300 µl droplets of medium under oil in a ∆T Culture Dish System (Bioptechs Inc. Butler, PA, USA; see Van Blerkom et al., 1995) and observed continuously at 37° C for up to 2 h. When detected, motile protrusions from the surface of cumulus cells were recorded on videotape with image capture and processing on a Macintosh 6100/60AV Power PC using Fusion Recorder and Adobe Photoshop software. Time-lapse studies were performed over several days with image recordings made on optical disk and processed as indicated above. Most cultures were maintained for ~3 months and examined microscopically during each scheduled media change. Immunofluorescence analysis of growth factors in cumulus and granulosa cell cultures Granulosa and cumulus cells were grown on cleaned, untreated glass coverslips in Dulbecco’s modified Eagle’s medium (DMEM)/F10 (50/ 50) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA, USA) and 10 µg/ml gentamicin (Gibco). Prior to fixation, cells were washed three times with phosphate-buffered saline (PBS) at 37°C. Cells were fixed in PBS containing 3.7% formaldehyde for 20 min at room temperature. After fixation, cells were permeabilized by incubation in a detergent solution containing 0.1% Triton X-100 and 0.1% Nonidet P-40 (Sigma, St Louis, MO, USA) in PBS for 20 min at room temperature. Permeabilized cells were placed in a PBS solution containing 1% bovine serum albumin (BSA) and incubated at 4°C for 1 h. Fixed and permeabilized cells were transferred to a fresh solution of PBS containing 1% BSA for an additional hour prior to

antibody exposure, or were stored in this solution for several days at 4°C. Prepared cells were reacted with the following primary antibody solutions: leptin (Ob [Y-20]), TGFβ1 and TGFβ2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), VEGF (MAB293; R&D Systems, Minneapolis, MN, USA), bFGF (AB-3) and TGFα (AB-2) (Oncogene Research Products, Cambridge, MA, USA) as well as solutions containing non-specific, control immunoglobin molecules. All antibodies were used at a concentration of 1 µg/ml and were prepared in PBS with 2% BSA, with the exception of the VEGF antibody which was used at a concentration of 5 µg/ml. Non-specific control antibodies were used at the concentrations of their specific counterparts. Primary antibody incubations were done at 4°C for 8 h or overnight. After 2 post-primary antibody washes in PBS containing 1% BSA, cells were incubated in PBS containing 10% serum of the species of the secondary antibody for 1–2 h at room temperature to block non-specific, secondary antibody binding. Cells were washed a final time in a solution of PBS containing 1% BSA prior to the secondary antibody reaction, which persisted for 2 h at 4°C. Secondary antibody solutions were prepared as follows: goat anti-rabbit antibody, fluorescein isothiocyanate (FITC)-conjugated or biotinylated, 1:200 dilution (Sigma), and goat anti-mouse antibody–FITC conjugate, 1:150 dilution (Sigma), both in PBS at 2% BSA. After immunostaining, coverslips were washed twice with a solution of PBS containing 1% BSA and were then incubated in 10 drops of Slowfadelite equilibration buffer-component C (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature prior to mounting onto slides in a drop of Slowfade-lite component A (Molecular Probes). Coverslips reacted with biotinylated secondary antibody were incubated further in a 1:100 dilution of streptavidin–Texas Red (Molecular Probes) prepared in PBS at 2% BSA for 1 h at room temperature. After reaction with the fluorochrome, coverslips were processed as described above before mounting onto slides. In some instances cells were double-stained to determine whether a single TS contained more than one factor. Procedures for fixation and analysis of thin sections by transmission electron microscopy and fluorescence examination of antibody-stained cells by scanning laser confocal microscopy have been described previously (Van Blerkom et al., 1995).

Results Light microscopic characteristics of tethered structures Detailed light microscopic examination of cultured human cumulus oophorus and granulosa cells obtained from preovulatory follicles revealed a remarkable and unusual morphodynamic activity characterized by multiple, balloon-like structures tethered to the cell surface (Figures 1–5). The dynamic nature of these TS was evident during real-time observation and involved a rate of formation of the order of minutes. Time-lapse images of a single cumulus cell on which multiple TS developed, detached or were resorbed during a 53 min period of observation are shown in Figures 6–14. Although this pattern was representative of most cumulus and granulosa cells that exhibited this activity, in some cells the evolution of TS was extremely rapid (,1 min). The following results derived from time-lapse analysis performed over several days suggested that this cellular activity may be episodic or cyclical: (i) intense periods of TS formation lasting ~8–12 h abruptly ceased with either resorption or detachment of the TS, (ii) a period of apparent inactivity lasting ~12–18 h ensued, followed by (iii) a sudden resumption of TS development. TS originated from multiple cytoplasmic loci associated with 2227

M.Antczak, J.Van Blerkom and A.Clark

Figures 1–5. Figure 1 is a light microscopic image of tethered structures (TS) (arrows) emanating from a cultured granulosa cell. Figures 2–5 are light microscopic views of a TS (arrow) originating from a mass of newly aspirated human cumulus oophorus. These images were taken at 20 s intervals and show lateral movement of the TS (inset). Original magnifications: Figure 1, 3500; Figures 2–5, 3140; insets, 3315.

aggregates of membranous structures (see below). Although most TS were balloon-like at some time during their existence, the dynamic nature of these elements was indicated by continual changes in size and shape that often occurred within seconds. When fully formed, TS often exhibited a rotary motion that originated from the apical surface of the bulbous projection. In time-lapse images, small vesicular elements were observed to move from the basal to the apical portions of the stalk and to disappear as discrete entities detectable by light, electron, or immunofluorescence microscopy (see below) as they progressed to the more apical portions of the TS. Prior to detachment, some TS displayed a sudden onset of plasma membrane blebbing (Figures 8c, 12f) at the basal portion of the tether close to the site of attachment to the plasma membrane. After TS detachment, the bulbous portion was often observed on the cell surface (Figure 22, small arrow), occasionally with a small portion of the tether still attached (Figure 22, large arrow). These detached structures retained motility as indicated by changes in shape and by an apparent ability to migrate across the cell surface. The possibility that an actin-based system provided the motility involved in TS formation and movement before and after detachment 2228

was supported by inhibitor studies. Within 1–3 min after the addition of cytochalasin D at a concentration of 10 µg/ml, TS formation ceased and existing structures were resorbed into the cytoplasm. The spontaneous detachment of the spherical portions of the TS from cells grown in culture is suggestive of a secretory activity. TS were identified in newly aspirated cumulus oophorus and in sheets of granulosa cells displaced from the follicular wall during aspiration, and were mechanically dispersed into small fragments. TS-like elements from these sources were isolated for analysis at high magnification and were recorded and shown to exhibit lateral movements over intervals of several seconds (Figures 2–5). The detection of structures in newly aspirated cumulus and granulosa cell masses that have morphodynamic characteristics virtually identical to those of TS in cultured cells suggests that this activity exists in the pre-ovulatory follicle. The detection of TS during the first few days of culture required very close inspection as their presence could be easily mistaken for extracellular debris. The relative proportion of cultured granulosa and follicle cells involved in TS formation appeared to be patient-specific (pooled) and, for cumulus cell cultures established from individual follicles, follicle-specific as well. For most patients, ~10% or less of their cells displayed TS formation during multiple inspections over months of culture. However, the relative abundance of these cells was higher for some patients, and in particular for three patients who experienced hyperstimulation syndrome after IVF–embryo transfer. For these patients, it was estimated that .50% of the cultured granulosa and cumulus cells were involved in TS formation both from the outset of culture and for months thereafter. Subcellular characteristics of tethered structure formation Serial thin section reconstruction of TS-producing granulosa and cumulus cells revealed a remarkable subcellular organization characterized by compartmentalization of the cytoplasm from which TS arose. The TS-forming region of the cell (Figure 15, asterisk) was densely packed with cisternae, small vesicles, and vacuoles, and was clearly separated from the nuclear compartment by a dense filamentous network (Figure 15, arrows). Mitochondria, cisternae of rough-surfaced endoplasmic reticulum, lipid inclusions, and polyribosomes were confined to the nuclear compartment (Figures 15 and 16). At higher magnification, this internal network was an apparently continuous layer of filaments with periodic, electron-dense regions (Figures 16 and 21). The presence of small foci of cytoplasmic continuity between the two compartments suggests that the filamentous network is porous. The apical plasma membrane associated with TS formation exhibited a thick layer of subplasmalemmal microfilaments (Figures 15 and 16, arrowheads), which at higher magnification was characterized by closely spaced, electron-dense tubular structures oriented perpendicular to the plasma membrane (Figure 20, arrows). This membrane was covered by a glycocalyx (Figure 20, arrowheads). In contrast, a very thin layer of microfilaments was associated with the subplasmalemmal cytoplasm on the basal or non-tether-producing portion of the cell. The first phase of TS formation was the emergence of a small surface


Figures 6–14. Time-lapse light microscopic images showing eight tethered structures (TS) (a–h), and demonstrating their formation (e, f, g, h), motility, and disappearance (a, b, c, d), that occurred on a single cultured granulosa cell during at 53 min interval. After detachment from the cell, the bulbous portion of TS designated ‘c’ in Figure 9 remained on the cell surface for several minutes. Detachment is often preceded by intense blebbing of the plasma membrane at the basal portion of the stalk (Figures 7 and 8, arrowheads). Times (h, m, s) are presented in the lower left-hand corner of each photograph. Original magnifications: Figures 6–14, 3450.

protrusion with a core of densely packed vesicles and cisternae (Figures 16 and 19, large arrowhead). The protrusion was surrounded by a dense outer layer of filaments similar in appearance to those present in the apical subplasmalemmal cytoplasm (Figure 16, white asterisks; Figure 17). Small cytoplasmic blebs, some of which appeared to be budding from the plasma membrane, developed on the lateral and apical surfaces of the protrusion(s). The blebs contained a homogeneous filamentous material (Figures 16 and 19, asterisk) with fine structural characteristics usually associated with microfilaments. These protrusions rapidly extended into a fully-formed TS. A representative image of a fully-developed TS observed

in thin section is shown in Figure 17. Typically, TS were organized into the following three compartments: (i) a basal section continuous with the underlying cytoplasmic compartment and densely packed with small vesicles, cisternae and electron-translucent vacuoles (Figure 17, A); (ii) a stalk-like structure or tether of relatively high electron density that was usually free of the type of membranous structures located in the basal region (Figure 17, B); (iii) a bulbous structure of moderate electron density (Figure 17, C) that contained a homogeneous, amorphous (i.e. non-filamentous) material devoid of any vesicular or vacuolar elements (Figure 18). In some TS, a layer of electron-translucent vacuoles was present at the boundary between the C and B regions (Figure 17, 2229


Figures 15–21. Representative transmission electron microscopic images of tethered structure (TS) formation in cultured human granulosa cells. Figure 15 shows the unusual organization of a TS-producing cell during the earliest stage of TS development. The secretory portion of the TS-producing cell (asterisk) is characterized by a dense accumulation of vesicles, vacuoles (v) and cisternae which is clearly separated from the nuclear (N) compartment by a dense filamentous network (Figure 15, arrows) that extends to the plasma membrane. Periodic, electron-dense aggregates are observed within this internal network at higher magnification (Figures 16 and 21, arrows). The nuclear compartment contains mitochondria (M), rough-surfaced endoplasmic reticulum (RER) and polyribosomes and lipid inclusions (Figures 15 and 16). Subjacent to the plasma membrane on the apical or TS-forming portion of the cell is a dense network of microfilaments (arrowheads) which at higher magnification (Figure 20) is observed to contain periodic elements aligned perpendicular to the plasma membrane (Figure 20, white arrows). In contrast to the basal cell surface, the apical plasma membrane is covered by a glyocalyx (Figure 20, arrowheads). As the TS forms, the size of the secretory compartment diminishes (Figure 16, asterisk) as the vesicular and cisternal elements enter the protrusion. Small structures containing a homogeneous filamentous material (Figure 16, black asterisks) arise from the surface of the emerging TS. The wall of the early TS contains a dense filamentous material that appears to be actin-based (Figure 16, white asterisks). Figure 17 is a representative electron micrograph of a fully-formed TS showing its three characteristic compartments: (A) a basal region packed with vesicles and cisternae, (B) a mid region free of secretory elements and enclosed by a thick subplasmalemmal filamentous layer, and (C) a bulbous or balloon-like terminus that has a homogeneous texture (Figure 18, higher magnification) and is free of any cytoplasmic structures or organelles. Small vacuoles are often present at the boundary between the B and C compartments (Figure 17, arrowheads). At higher magnification (Figure 19), the dense packaging of vacuoles (v) and cisternae (c) and enclosure of these elements by a thick filamentous wall (asterisks) is clearly evident in cross-sectional views of the basal (A region) of the TS. Original magnifications: Figure 15, 36570; Figure 16, 313 000; Figure 17, 31600; Figure 18, 34400; Figure 19, 314 600; Figure 20, 39500; Figure 21, 328 000.

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arrowheads). In comparison to compartments A and B, the apparent thickness of subplasmalemmal microfilaments was reduced considerably in compartment C (Figure 20, arrows). Identification of growth factors in tethered structures TS-forming cells were probed by immunofluorescence for the following growth factors known to be synthesized by human granulosa and cumulus cells: bFGF, TGFα, TGFβ1, TGFβ2, VEGF and leptin. Only VEGF-, leptin- and TGFβ2-associated immunofluorescence was detected in TS (Figures 25–30). Changes in the intensity of immunofluorescence for these factors during the progressive growth of a representative TS is shown in Figures 25 and 26 (both 3900 magnification). Potential sites of TS formation in cultured cumulus and granulosa cells initially appeared under differential interference contrast optics as large vacuolar structures (Figures 22–24, arrowheads). With continual observation focused on these regions, some of these structures began to protrude from the cell surface (Figure 23, arrow) and within minutes evolved into a TS (Figure 24, asterisk). Anti-leptin, anti-VEGF, and anti-TGFβ2 immunofluorescence was detectable throughout the protruding region of the cytoplasm (Figure 25, asterisk) but as the TS developed, antibody fluorescence was concentrated in the balloon-like compartment (Figure 26, asterisk). Figure 27 is a representative scanning laser confocal image of a granulosa cell from which multiple TS emerged that were immunostained both for VEGF (Figure 28) and leptin (Figure 29). Computer processing of these digital images permitted 90° rotation such that the elevation of the TS from the cell surface was evident (Figures 28 and 29). The relative intensity of immunofluorescence associated with these factors within individual TS varied between TS-forming cells. For example, some TS exhibited intense leptin fluorescence but little VEGF fluorescence, and vice versa. Figure 30 is a representative image of a negative imunofluorescence control and TS probed for TGFα, bFGF and TGFβ1. Discussion The presence of a wide array of growth factors, mitogens, and cytokines in ovarian follicular fluid reflects the many different developmental functions of follicle cells during the pre- and post-ovulatory stages. After ovulation, for example, TGFβ stimulates progesterone production by granulosa cells (Magoffin et al., 1989), while TNF-α, EGF, bFGF, IL-1, IL-6, and colony stimulating factor-1 may be involved in proliferation and the specialized functions(s) of granulosa cells and the proliferation of fibroblasts and epithelial cells at the ruptured follicular apex and stigma (Ziltener et al., 1993; Brannstrom et al., 1994). VEGF is secreted by cumulus and granulosa cells and accumulates to relatively high concentrations in preovulatory follicular fluid (Van Blerkom et al., 1997). VEGF produced within each follicle may promote the development of the perifollicular micovasculature (Ravindranath et al., 1991; Van Blerkom et al., 1997) whereas after ovulation the continued synthesis of VEGF by residual granulosa cells has been suggested to promote angiogenesis within the developing corpus luteum (Kamat et al., 1995). An over-expression of

Figures 22–26. Representative differential interference contrast images of tethered structure (TS)-forming granulosa cells are shown in Figures 22–24. The black arrowheads indicate an early stage of TS evolution characterized by spherical protrusions from the cell surface. The bulbous portion of a detached TS on the cell surface (long arrow) along with a remnant of the plasma membrane stalk (black triangle) is shown in Figure 22. Figures 25 and 26 are scanning laser confocal immunofluorescence images of TS-forming cells stained with anti-vascular endothelial growth factor (VEGF). The intensity of fluorescence increases and moves progressively as the TS develops from a cell surface protrusion (Figure 25, asterisk) to a fully-formed TS (Figure 24, black and white arrows) such that virtually all anti-VEGF, -leptin, or -transforming growth factor-β2 staining was concentrated in the balloon-like compartment (asterisk, Figure 26). Original magnifications: Figure 22, 3220; Figures 23 and 24, 3440; Figures 25 and 26, 3660.

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Figures 27–30. Computer-generated scanning laser confocal pseudo-colour images of tethered structure (TS)-producing granulosa cell double-stained with anti-vascular endothelial growth factor (VEGF) [fluoroscein isothiocyanate (FITC) secondary] and anti-leptin (biotinylated secondary and strepavidin-Texas Red tertiary). Figure 27 is a surface view showing the bulbous portion of the TS stained with anti-VEGF whereas Figure 28 displays the same image rotated 90° to demonstrate the elevation of the TS above the cell surface. Figure 28 demonstrates the pattern of anti-VEGF fluorescence in the FITC channel and Figure 29 demonstrates the pattern of anti-leptin fluorescence in the rhodamine channel. Note the relative reduction in the intensity of anti-VEGF fluorescence and in the TS on the far right in Figure 28. Figure 30 is a representative scanning confocal image of a negative control and of those TS-producing cells probed with antibodies directed against the other growth factors described in the text. In pseudo-colour images, the intensity of antibody fluorescence is related to a continuous colour spectrum with blue and white at the low and high ends respectively, as indicated on the far right-hand corner of Figure 30.

VEGF has been implicated in the abnormally high level of fluid leakage from the follicular vasculature that characterizes ovarian hyperstimulation syndrome (OHSS) (McLure et al., 1994). We have recently demonstrated that leptin, a protein growth factor thought to be an adipocyte product involved in the regulation of body fat deposition (Considine et al., 1996) is synthesized by pre-ovulatory human cumulus oophorus and granulosa cells (Cioffi et al., 1997). Although the function of follicular-derived leptin has not been determined, roles in oocyte and early embryo transcriptional regulation (Antczak and Van Blerkom, unpublished), ovarian steroidogenesis (Chehab et al., 1997), and ‘preparation’ of the endometrium for implantation have been suggested (Cioffi et al., 1997). We describe the occurrence in human granulosa and cumulus oophorus of a highly specialized cell type that accumulates and sequesters VEGF, leptin, and TGFβ2 in membrane-bound structures and releases these ‘packets’ of growth factors by means of an unusual apocrine-like mechanism. While other cell types have well-described aprocine secretion, to the 2232

best of our knowledge, cells with this type of intracellular organization and similar morphodynamic characteristics have not been reported previously. With respect to gross morphology, however, TS resemble some of the pleiomorphic cytoplasmic protrusions elaborated within the mammalian reproductive tract such as (i) apocrine cytoplasmic projections on the surface of human Fallopian tube secretory cells (Ferenczy and Richart, 1974) and (ii) endocytotic structures (pinopods) (Enders and Nelson, 1973; Nikas et al., 1995), and putative exocytotic projections (Van Blerkom and Motta, 1979) that occur on the luminal surface of non-ciliated uterine epithelial cells. Our studies show that a subset of ovarian follicles cells form balloon-like structures tethered to the cell surface by means of a slender stalk of plasma membrane. Light microscopic examination of newly aspirated cumulus oophorus masses and granulosa cells indicates a pattern of cellular activity with morphodynamic characteristics that is virtually identical to TS formation observed in culture. In cumulus and granulosa cell masses examined immediately after follicular aspiration, pre-


existing TS-like elements exhibited motion independent of the underlying cell(s) (which were non-motile) from which they often detached during the period of examination. Time-lapse studies of cell cultures demonstrate that TS develop rapidly, with some cells producing TS in ,90 s. Although these structures can be stable for several hours, they can also be resorbed back into the cytoplasm or released from the cell surface. The rapid retraction of TS in the presence of cytochalasin D is indicative of a kinetic process that is actin-based. TS migrate across the cell surface prior to detachment and retain motility after detachment. This motile capacity may be supported by subplasmalemmal microfilaments identified by electron microscopy in the different TS compartments. The ability of TS to remain motile after release may play a role in extending the effective range of TS-producing cells beyond the scope of their immediate environment. This capability would be particularly useful in a closed system devoid of vascular elements, such as the pre- and early post-ovulatory follicle. The rotary motions typically displayed at the apex of the bulb of the TS may represent an activity designed to facilitate movement of the bulb between cells and through the extracellular matrix. In this regard, TS have been observed to extend laterally along the surface of the dish as well as beneath the surface of adjacent cells (data not shown). A secretory function is suggested by the morphodynamics of TS generation and by the very unusual organization of the cytoplasm from which these structures emerge. Reconstructions of serial thin sections demonstrate that the cytoplasmic compartment from which TS develop is densely packed with vacuoles, vesicles, and tubular cisternae. This compartment is separated from cytoplasmic organelles such as mitochondria, polyribosomes, lipid inclusions, and rough-surfaced endoplasmic reticulum by a dense, net-like array of cytofilaments. It is likely that this intracytoplasmic organization prevents these organelles from entering the secretory compartment and being lost to the TS. In the same respect, the dense accumulation of membranous structures in the vesicular portion of these cells may reflect storage of membrane precursors required for rapid expansion of the plasma membrane during intense periods of multiple TS formation. Perhaps a phase of relative inactivity is required to replenish these putative precursors. The present findings indicate that leptin, VEGF, and TGFβ2 are packaged into vesicles within the ‘secretory’ compartment of these specialized cells, and that these vesicles migrate or are transported to the TS-forming region and enter the emerging cytoplasmic projection. Although a specific role of TS formation by ovarian follicle cells has not been determined, we propose that this activity may represent a novel process by which the specific growth factors can be accumulated at high concentration, sequestered in discrete membrane-bound structures, and subsequently discharged in a directed and regulated manner. TS formation is observed to have an episodic property characterized by phases of intense multiple TS formation followed by periods of relative dormancy. All the evidence obtained in this study supports the premise that TS-producing cells are long-lived and intermittently engage in TS activity over protracted periods of time. The identification of structures

closely resembling TS in newly isolated granulosa and cumulus masses makes it unlikely that TS formation in vitro is an aberration or culture artefact. Further, the very nature of the phenomenon, its persistence in the same cultured cells for months, the unusual level of activity, and the absence of light or electron microscopic evidence of membrane, cytoplasmic, or nuclear disorganization or deterioration indicate that TS formation is a specialized activity of a subpopulation of follicle cells and not a manifestation of some form of cell death such as apoptosis. Our previous studies have shown that VEGF occurs in preovulatory human follicle fluid at relatively high concentration (Van Blerkom et al., 1997) and that leptin levels in this fluid increase after the administration of an ovulatory dose of human chorionic gonadotrophin (Cioffi et al., 1997). The elaboration of TS may be one means by which the levels of leptin, VEGF and TGFβ2 are regulated during follicle growth and preovulatory development. If retained as membrane elements within the granulosa or residual antral fluid, they could provide a reservoir of growth factors to be utilized after ovulation. Perhaps qualitative and quantitative changes in gonadotrophins within the pre- and post-ovulatory follicle alter the type of factors accumulated by these cells and the intensity or frequency of TS formation. In this respect, the presence of TS in an intact or detached form within the residual mass of granulosa cells could provide locally high concentrations of VEGF to promote angiogenesis during the early stages of corpus luteum formation. Although leptin is one of the growth factors contained within TS, a specific role(s) for this protein in the biology of the pre- or post-ovulatory follicle has not been determined (Cioffi et al., 1997). The frequency with which TS-forming cells occur in granulosa cell cultures established from individual patients may be of clinical relevance. Observations of granulosa and cumulus oophorus cell cultures indicated that over half of the granulosa cells from three women who developed severe OHSS after IVF–embryo transfer produced TS. This finding is in contrast to the 5–10% frequency of TS-forming cells observed for most cultures established from patients included in this study. Whether the elevated concentration of VEGF detected in the serum of women experiencing OHSS (McLure et al., 1994) correlates with an abnormally high frequency of TS-forming granulosa cells that can accumulate and secrete VEGF as described here is currently under investigation. In summary, our findings demonstrate that a novel method of VEGF, leptin and TGFβ2 accumulation, packaging and release occurs in a highly specialized population of human ovarian granulosa and cumulus cells. Although we were unable to detect the presence of TGFα, bFGF and TGFβ1 in TS, this may be a consequence of our particular cell culture system and we cannot preclude the possibility that these factors may be found in TS under different in-vitro conditions. TS formation may contribute to the accumulation of a variety of known, and possibly other as yet unknown factors in the pre-ovulatory follicle, and may provide locally high concentrations in a stored form that could be utilized for post-ovulatory processes such as follicular repair, steroidogenesis, and angiogenesis in the early corpus luteum. TS-forming cells as described in this 2233


study have also been identified in murine follicle cell cultures suggesting that this activity may be a general phenomenon in the mammalian ovarian follicle (M.Antczak and J.Van Blerkom, unpublished findings). The extent to which TS production is patient or follicle specific, and whether other growth factors or cytokines detected in human follicular fluid can also accumulate by this mechanism, remain to be determined. Acknowledgement We thank Cathy Van Blerkom, MD for her comments on the manuscript, and Drs Kenneth Faber, Samuel Alexander, and George Henry for their clinical contributions. This work was supported by a grant from the National Institutes of Child Health and Human Development, National Institutes of Health (HD-31907).

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