THE ANATOMICAL RECORD 233:41-52 (1992) Immunoelectron Microscopic Localization of Laminin in Rat Ovarian Follicles VIJITTRA LEARDKAMOLKARN AND DALE R. ABRAHAMSON Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand (V.L.) and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama ABSTRACT We studied the immunohistochemical and ultrastructural distribution of laminin in ovaries of immature and mature rats. When sections from 1-8-week-old rat ovaries were labeled directly with conjugates of affinity purified anti-laminin IgG-horseradish peroxidase (HRP), the antibodies bound to all ovarian basement membranes including those surrounding follicles in different stages of maturation. In addition, intracellular labeling was seen in granulosa and theca cells of follicles undergoing rapid development (preantral and antral stages) and in basement membrane-like structures of the Call-Exner bodies. Intracellular laminin was generally not detected, however, in any cells of primordial or atretic follicles. Tissue processed for immunoelectron microscopy 1 hour after the intravenous injection of anti-laminin IgG-HRP showed binding of antibody in linear patterns along endothelial and follicular epithelial basement membranes. Discontinuous strands of laminin-positive, extracellular matrices were also seen between theca cells of all follicles. In addition, injected anti-laminin IgG labeled perisinusoidal basement membranes located within corpora luteae and patches of basement membrane material between granulosa lutein cells. When ovaries were examined 5 d after the intravenous injections of anti-laminin IgG-HRP, uneven or segmented labeling was found in subepithelial basement membranes surrounding developing follicles. Our results therefore indicate that granulosa and theca cells participate directly in basement membrane laminin biosynthesis and suggest that this new laminin is spliced into existing basement membranes during follicular growth. o 1992 Wiley-Liss, Inc. Follicular growth in the mammalian ovary leads to oocyte maturation and is accompanied by proliferation and differentiation of the cellular components of the follicular wall, which consists of internal granulosa (epithelial) and external theca (stromal) layers. Generally, these two layers are separated by a follicular basement membrane that ultrastructurally resembles basement membranes found commonly throughout the body (Anderson et al., 1978; Bjersing and Cajender, 1974; Peters and McNatty, 1980). In addition, immunohistochemical studies carried out at the light microscopic level have also documented the presence of collagen type IV, laminin, and heparan sulfate proteoglycans within ovarian follicular basement membranes (Bagavandoss et al., 1983; Palotie, et al., 1984; Wordinger et al., 1983). In the rat, primordial follicles start development during the neonatal period when resting oocytes enlarge and an associated single layer of granulosa cells begin to proliferate (Harrison and Weir, 1977). As growing follicles swell and progress through antral stages, the circumference around the follicular wall and basement membrane increases several times prior to disruption at ovulation (Espey, 1974; Espey and Coons, 1976; Peters and McNatty, 1980).The time interval for follicle maturation, as measured from primordial to ovulatory stages, is relatively rapid in rats and lasts approxi0 1992 WILEY-LISS, INC. mately 18-20 days (Hage et al., 1978; Harrison and Weir, 1977). In the present study, we investigated the ultrastructural distribution of laminin in follicular basement membranes of immature and mature rats. In addition, we sought to determine which cells are responsible for basement membrane laminin formation during ovarian follicle growth. MATERIALS AND METHODS Animals and Reagents Nineteen female Sprague Dawley rats, ranging in age from 1 day to 2 months old, were used in these experiments. Sera from laminin-immunized sheep and rabbits were collected and anti-laminin specific IgGs were affinity isolated as described previously (Abrahamson and Caulfield, 1982; Abrahamson, 1985; Abrahamson and Caulfield, 1985). Control sheep and rabbit IgGs, chromatographically pure, were purchased (Cappel-Organon Teknika Corporation, Durham, NC). Sheep and Received August 6, 1991; accepted October 21, 1991. Address reprint requests to Dr. Dale R. Abrahamson, Department of Cell Biology, University of Alabama at Birmingham, UAB Station, Box 302, Birmingham, AL 35294-0019. 42 V. LEARDKAMOLKARN AND D.R. ABRAHAMSON Table 1. Age and number of rats processed for laminin immunohistochemistry 1d 1 week Aae 3 weeks 1 1 2 1 1 1 In vitro, post-fixation labeling Anti-laminin IgG Control In vivo, pre-fixation labeling’ Fixation 1 h post-anti-laminin injection Control Fixation 5-6 d post-anti-laminin injection Control 1 2 1 1 4 weeks 8 weeks 3 1 1 1 1 1 ‘Ages shown are ages at time of injection. rabbit anti-laminin and control IgGs were conjugated directly to horseradish peroxidase (HRP) (Type VI, Sigma Chemical Company, St. Louis, MO) using methods described by Nakane and Kawaoi (1974). lmmunohistochemical Labeling Procedures For in vivo, pre-fixation labeling, rats were anesthetized with ether and they then received intravenous injections of sheep anti-laminin IgG-HRP (-1.0 mg IgG/lOO g body weight) via the saphenous vein. One hour or 5-6 days after the injection, ovaries were removed, fixed for 2 hours by immersion in Karnovsky’s fixative (Karnovsky, 1965), and processed histochemically for peroxidase (Graham and Karnovsky, 1966) a s described before (Leardkamolkarn and Abrahamson, 1988; Leardkamolkarn et al., 1989, 1990). Tissue was then post-fixed in 2% osmium, routinely dehydrated with ethanol and propylene oxide, and embedded in epoxy resin. Sections 1 pm thick were stained with toluidine blue for light microscopy. For electron microscopy, ultrathin sections (70 nm thick) were stained briefly with lead citrate (Reynolds, 1963) and examined with a n accelerating voltage of 60 or 80 kV in a JEOL 100 CX electron microscope. Rats serving as controls received normal sheep IgG-HRP and tissues were processed in the same procedure. For in vitro, post-fixation labeling, ovaries were removed from uninjected rats and fixed by immersion in 2%paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 2 hours. Sections, 30 pm thick, were obtained with a Vibratome and incubated with rabbit anti-laminin IgG-HRP or control rabbit IgG-HRP (200 pg/ml) for 48 hour and further processed for peroxidase histochemistry as described above. The numbers and ages of rats used in the separate experiments are shown in Table l. RESULTS Laminin Distribution in Immature Rat Ovary Ovaries of newborn and 1-week-old rats contained follicles in almost all different stages of maturation including primordial, primary, secondary, pre-antral, and antral follicles. One hour after the intravenous injection of sheep anti-laminin IgG-HRP, peroxidase reaction product was seen in all follicular and capillary basement membranes (Fig. 1).In addition, laminin immunoreactivity was scattered between theca cells surrounding pre-antral follicles (Fig. 1).Electron microscopy showed a dense, linear deposition of anti-laminin Fig. 1 . Phase-contrast micrograph of ovary section from a l-weekold rat, 1h after receiving an intravenous injection of affinity purified anti-laminin IgG conjugated to HRP. Note that the peroxidase reaction product is present in a linear pattern in all follicular basement membranes (arrowheads).There is also some scattered HRP reaction product between developing theca cells (arrows). x 1,150. IgG-HRP in a continuous basement membrane underlying follicular epithelial and capillary endothelial cells (Fig. 2). In contrast, discontinuous fragments of LAMININ LOCALIZATION IN RAT OVARIAN FOLLICLES 43 Fig. 2. Electron micrograph of ovary from a 1-week-old rat, 1 h after receiving an intravenous injection of sheep anti-laminin IgG-HRP. A continuous or linear deposition of HRP reaction product can be seen along capillary endothelial (single arrows) and follicular epithelial basement membranes (arrowheads). In addition, fragments of laminin-positive, basement membrane-like material are dispersed between the forming theca interna cells (double arrows). Gr, granulosa epithelial cell; CL, capillary lumen; Th, theca cell. x 13,000. laminin-positive, basement membrane-like matrices were found between forming theca cells lying close to the follicle (Fig. 2). To determine which cells participate in ovarian laminin biosynthesis, sections of ovaries from uninjected immature rats were labeled in vitro with rabbit antilaminin IgG-HRP. Dense laminin immunoreactivity was seen linearly along all follicular and vascular basement membranes (Figs. 3 and 4).Intracellular labeling was not seen within the follicular epithelial cells or oocytes of primordial follicles (Fig. 3). Laminin immunoreactivity was detected, however, within biosynthetic organelles of granulosa epithelial cells in primary and secondary (antral) follicles (Fig. 4).Occasionally, laminin positive material was also detected by light microscopy as a round or oval vesicle inside the growing follicles (Fig. 5a). Electron microscopy showed that this vesicle was situated extracellularly among the granulosa cells, thereby corresponding to structures referred to a s “Call-Exner bodies” (Fig. 5b). In addition, intracellular laminin was identified within the rough endoplasmic reticulum of developing theca cells adjacent to growing follicles (Fig. 4). Intracellular laminin immunoreactivity was not found in epithelial or theca cells of degenerating follicles and there was no labeling whatsoever on sections treated with control IgG. Five days after the intravenous injection of sheep anti-laminin IgG-HRP into immature rats, discontinuous lengths of HRP-reaction product were found along the follicular basement membranes of growing follicles at various stages of development (Fig. 6 4 . Ultrastructural examination also showed a clear fragmentation of peroxidase reaction product in the basement membranes underneath granulosa epithelial cells of maturing follicles and well-reacted regions were interspersed between segments with weak or no peroxidase activity (Fig. 6b). Based on our interpretation in developing kidney glomeruli and developing adrenal and pituitary glands (Abrahamson, 1985; Leardkamolkarn and Abra- 44 V. LEARDKAMOLKARN AND D.R. ABRAHAMSON Fig. 3. Electron micrograph of primordial follicle from l-month-old rat ovary. Tissue was fixed in 2% paraformaldehyde and 0.05% glutaraldehyde and Vibratome sections were labeled directly with affnity purified anti-laminin IgG-HRP. Dense laminin immunoreactivity is seen linearly along the follicular basement membrane (arrowheads) but not within the cytoplasm of the follicular epithelial cells (Ep). 00, oocyte; arrowhead, follicular basement membrane. x 7,000. hamson, 1988),we believe that the unlabeled segments represent basement membrane assembled after the intravenous injections were given. in non-ovulated follicles undergoing early degenerative changes, non-linear laminin immunoreactivity was found underneath granulosa cells (Fig. 9). The extracellular matrices in the theca layers of these folli- Distribution of Laminin in Mature Rat Ovary As compared to those of immature rats, sexually mature ovaries contained greater numbers of developing antral and pre-ovulatory follicles and there were also increased corpora luteae formation. The distribution of laminin, as determined by the binding of intravenously injected HRP-conjugated antibody, was generally similar to that seen in immature ovaries, however. One hour after injection, anti-laminin IgG-HRP was bound in linear patterns to the epithelial basement membrane around follicles, and to strands of basement membrane-like material in the theca layers as well as the corpora luteae (Figs. 7 and 8). The theca layers were much more organized than those seen in young rats, and the extracellular strands of laminin-positive matrix were also more extensive (Fig. 8). In addition, Fig. 4. Electron micrograph of ovarian follicle during rapid growth stage. Tissue was taken from l-month-old rat, fixed and labeled as in Figure 3. Laminin immunoreactivity is detected not only in the follicular basement membrane but also intracellularly within the rough endoplasmic reticulum (RER) of granulosa cells (arrows). Positive intracellular labeling is also seen in the cytoplasm of a fibroblastic cell in areas of theca formation adjacent to the follicle (arrowheads). Gr, granulosa epithelial cell. x 11,000, Fig. 5. Phase-contrast micrograph (a)and electron micrograph (b)of ovary sections from maturing follicles. Tissues were fixed and labeled as in Figures 3 and 4. Dense laminin immunoreactivity is seen in Call-Exner bodies between granulosa cells (arrows).00,oocyte. Laminin immunoreactivity is also seen intracellularly (*) within granulosa cells (Gr). a, x 2,200; b, x 9,000. Figs. 4 and 5. 46 V. LEARDKAMOLKARN AND D.R. ABRAHAMSON Fig. 6. Phase-contrast micrograph (a)and electron micrograph (b)of I-week-old rat ovary, 5 days after receiving a n intravenous injection of anti-laminin IgG-HRP. a: HRP reaction product is present (arrows) and absent (arrowheads) along the basement membranes of follicles at various stages of development (compare with Fig. 1). x 1,150. b: Lengths of well-reacted basement membranes (arrows) are interspersed with lightly to non-reactive lengths (bracket). The non-reactive areas probably represent newly formed segments of basement membrane that were added after the antibody injection was given. Gr, granulosa cells. x 12,500. cles also appeared to be undergoing remodeling in areas of theca cell differentiation (Fig. 9). In the welldeveloped corpora luteae where cells with characteristic steroid-producing morphology were seen, fragments of laminin-positive matrix were observed between the cells and in the subendothelium of developing sinusoids (Fig. 10). Five days after the injection of anti-laminin IgGHRP into mature rats, binding patterns in ovaries were very similar to those seen in immature rats processed in the same way. Basement membranes of the growing follicles were unevenly labeled but those of apparently quiescent follicles were labeled in continuous, linear patterns. and extend these earlier observations, and several new conclusions regarding ovarian basement membranes can now be made. First, granulosa epithelial cells of growing follicles, as well as stromal cells that probably correspond to theca precursors, synthesized laminin. Second, in both immature and mature rats, newly synthesized follicular basement membrane appeared to be inserted or spliced into existing matrix during follicular growth. Third, the in vivo labeling approach used here identified extensive basement membrane-like matrices located within corpora luteae. These results therefore demonstrate that basement membranes of cycling follicles undergo substantial and relatively rapid structural changes during pre- and post-ovulatory stages. Finally, since some reproductive disorders have been associated with autoimmune anti-laminin responses, the ability of circulating anti-laminin antibodies to bind ovarian basement membranes in vivo, which we document here, may represent a mechanism of immune injury. As others have observed previously, all pre-ovulatory ovarian follicles were surrounded by a distinct DISCUSSION Laminin has previously been immunolocalized by immunofluorescence (Wordinger et al., 1983) and peroxidase-anti-peroxidase light microscopic techniques (Palotie et al., 1984) to ovarian follicular basement membranes of mice and rats, respectively. Our present immunoelectron microscopic studies therefore confirm LAMININ LOCALIZATION IN RAT OVARIAN FOLLICLES Fig. 7. Phase-contrast micrograph of 2-month-old rat ovary, 1 h after the intravenous injection of sheep anti-laminin IgG-HRP. HRPreaction product is present in the basement membranes of a n antral follicle (arrows), and matrices of theca layers (Th), and corpus luteum 47 (CL). Note the absence of a continuous basement membrane around the corpus luteum (arrowheads).FA, follicular antrum; Gr, granulosa epithelial cells; CL, corpus luteum. X 1,150. basement membrane. Electron microscopy showed fur- served in growing ovarian follicles of most mammalian ther that, l h after injection, anti-laminin IgG-HRP species but, in general, these structures are only infreintensely labeled these structures throughout their full quently seen and their functions are not known. By widths in linear patterns that resemble those seen in light microscopy, Call-Exner bodies consist of rings or most other subepithelial basement membranes. Post- rosettes of granulosa cells distributed around small fixation labeling of immature and mature rat ovaries droplets of PAS-positive material (Motta and Nesci, also resulted in identification of laminin within biosyn- 1969) and early electron microscopic studies have thetic organelles of granulosa cells of growing follicles. shown that a continuous basement membrane layer These results therefore indicate that the follicular separates the granulosa cell rosette from the fluid conbasement membranes are assembled, a t least in part, tents (Motta, 1965). The intracytoplasmic labeling of by granulosa cells. Since some theca cells also con- laminin within granulosa cells further suggests that tained laminin immunoreactivity, the participation of these cells assemble the Call-Exner basement memtheca cells in synthesis of components destined for fol- branes, as well as those of the ovarian follicle proper. licular basement membranes is also possible, as sug- The presence of basement membranes within these structures may therefore represent nothing more than gested previously (Bagavandoss et al., 1983). The formation of the theca layers of the ovarian fol- an example of abnormal extracellular matrix secretion licular wall is not well understood. Previous investiga- by a cluster of misguided granulosa cells. Alternations have reported that, as primordial follicles begin tively, Call-Exner bodies may serve some physiological to grow, cells indistinguishable from fibroblasts align role and their basement membranes may therefore be themselves around the periphery of the granulosa important for orienting adherent cells. When ovaries of immature rats were examined 5 d layer, and these stromal cells then differentiate into theca cells. The strands of laminin positive, extracel- after the intravenous injection of anti-laminin IgGlular matrices that we observed in the theca layers HRP, uneven or segmented peroxidase reaction produndoubtedly provide structural support for the at- uct was found along basement membranes of growing tached cells. These unusual basement membranes are follicles. We interpret these results to indicate that the probably also synthesized by cells of the theca, as dem- unlabeled basement membrane segments were assemonstrated by the intracellular labeling for laminin that bled during the 5 d interval between in vivo labeling we observed in this location around growing follicles. and fixation. Further, the intermittent labeling patPost-fixation labeling of immature and mature ova- tern seen in basement membranes surrounding growries with anti-laminin IgG-HRP also labeled basement ing follicles is reminiscent of similar patterns seen in membranes within Call-Exner bodies in follicular expanding renal glomeruli (Abrahamson, 1985; Abragranulosa layers. Call-Exner bodies have been ob- hamson and Perry, 1986) and elongating tubules Fig. 8. Electron micrograph of 2-month-old rat ovary, showing higher magnification of area around preantral follicle 1 h after a n intravenous injection of sheep anti-laminin IgG-HRP. HRP reaction product is found continuously along the follicular basement membrane (large arrows). A thinner, subendothelial basement membrane around the ovarian microvasculature is also continuously labeled (small arrows). The theca layers, in contrast, contain discontinuous semi-concentric bands of laminin-positive, basement membrane-like material (arrowheads). Gr, granulosa epithelial cell, ThI, theca interna; ThE, theca externa. x 5,300. Fig. 9. Electron micrograph of 2-month-old rat ovary 1 h after the intravenous injection of sheep anti-laminin IgG-HRP. Shown in the upper part of figure is a portion of a mature, non-ovulated follicle undergoing degeneration and, in the lower part of figure, is the adjacent theca interna layer. Several gaps in HRP reaction product are found along the follicular basement membrane (large arrows). The subendothelial capillary basement membranes (small arrows) and thecal matrices (arrowheads) are also becoming reorganized (compare with Fig. 8). x 5,400. 50 V. LEARDKAMOLKARN AND D.R. ABRAHAMSON Fig. 10. Electron micrograph of corpus luteal cells from rat that received intravenous injection of sheep anti-laminin IgG-HRP. Laminin positive matrices are present in the perisinusoidal spaces (arrows) and between the granulosa luteal cells (arrowheads). L, lutein cells; S, capillary sinusoids. x 9,200. (Abrahamson and Leardkamolkarn, 1991) during kidney development. We believe, additionally, that these patterns seen in growing tissues represent the splicing of newly synthesized basement membrane into that already present and thereby provide a n expanded basement membrane surface area (Abrahamson, 1985; Abrahamson and Perry, 1986). An analogous splicing process has also been seen previously in developing adrenal and pituitary basement membranes (Leardkamolkarn and Abrahamson, 19881, as well as in those beneath the absorptive epithelium of intestinal villi (Trier e t al., 1990). Among the more striking differences between immature and mature ovaries were the increased abundance of maturing, pre-ovulatory follicles and corpora luteae in the latter. Injected anti-laminin IgG-HRP showed disruptions of the peripheral basement membranes in these pre- and post-ovulatory structures, as well as the appearance of basement membrane-like matrices located internally between granulosa lutein cells. The discontinuity of laminin immunoreactivity in these follicular basement membranes is in keeping with routine electron microscopic observations by others showing basal lamina disruption in follicles a t this stage LAMININ LOCALIZATION IN RAT OVARIAN FOLLICLES (Bjersing and Cajander, 1974; Cajander et al., 1984; Espey et al., 1981). The reasons for this basement membrane perforation are unclear but may be due to release of proteases by follicular wall cells in preparation for ovulation (Beers, 1975; Espey, 1974; Espey and Coons, 1976; Espey and Lipner, 1965). Projections of basal pseudopodia by granulosa cells through the basement membrane would also contribute to its disruption (Bjersing and Cajender, 1974; Cajender et al., 1984). Laminin has previously been identified by immunofluorescence microscopy within corpora luteae (Wordinger et al., 19831, and the immunoelectron microscopic images shown here identified laminin specifically within perisinusoidal areas and in basement membrane-like plaques between lutein cells. Basement membranes in the surrounding thecal layers of corpora luteae were also reorganized. The cellular components of the corpus luteum arise mainly from granulosa cells with contributions from the theca and surrounding stroma (Harrison, 1948). We therefore suspect that most of the extracellular matrices seen in corpora luteae originated during formation of the extensive sinusoidal vascular systems found in and around these post-ovulatory follicles. Such anastomosing networks of vessels bring each lutein cell into close contact with the vascular system (Bassett, 1943; Adams and Hertig, 1969) and this arrangement undoubtedly facilitates steroid hormone distribution. We did not observe any structural abnormalities or inflammatory cell infiltrates in ovaries of rats that received intravenous injections of heterologous antilaminin IgG under the conditions used in this study. Nevertheless, anti-basement membrane autoantibodies in humans can cause organ-specific autoimmune diseases including glomerulonephritis, Goodpasture’s syndrome (involving deposition of autoantibodies in alveolar as well as glomerular basement membranes), and bullous skin disorders (resulting in destruction of basement membranes in the dermal-epidermal junction) (reviewed in Timpl and Dziadek, 1986).Moreover, antibodies directed specifically against laminin can under certain experimental conditions mediate glomerular basement membrane damage in mice (Wick et al., 1982; Yaar et al., 1982) and rats (Abrahamson and Caulfield, 1982;Feintzig et al., 1986).In addition, antilaminin autoantibodies contribute to reproductive failure in monkeys and are toxic to cultured rat embryos in vitro (Carey and Klein, 1989; Weeks et al., 1989) and induce seminiferous tubule abnormalities in rat testes (Lustig et al., 1987). Our finding that apparently large amounts of circulating anti-laminin IgG bound to basement membranes of developing and mature ovarian follicles and interstitium therefore suggests that the ovary may be an additional possible target in autoimmune anti-basement membrane injury. ACKNOWLEDGMENTS These experiments were supported, in part, by NIH grant DK 34972 and an Established Investigatorship to D.R. Abrahamson from the American Heart Association. LITERATURE CITED Abrahamson, D.R. 1985 Origin of the glomerular basement membrane visualized after in viva labeling of laminin in newborn rat kidneys. J . Cell Biol., 100t1988-2000. 51 Abrahamson, D.R., and J.P. Caulfield 1982 Proteinuria and structural alterations in rat glomerular basement membranes induced by intravenously injected anti-laminin immunoglobulin G. J. Exp. Med., 156:128-145. Abrahamson, D.R., and J.P. 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