Apolipoprotein A-I production by chicken granulosa cells

^a The Department of Molecular Genetics, Biocenter and University of Vienna, Austria
^b Department of Biochemistry and Molecular Cell Biology, Biocenter and University of Vienna, A-1030 Vienna, Austria

	ABSTRACT

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In avian species such as the chicken, development of the oocyte is associated with massive deposition of yolk in this cell. Oocytes grow within the follicle, a compartment consisting of a very specialized set of cells and acellular structures. The oocyte is surrounded by the perivitelline layer and granulosa cells, which are separated from the thecae by a pronounced basement membrane. In addition to the production of yolk precursors in the liver, we have long implied that cells within the follicle make a direct contribution to the growth of the oocyte. Here we show that chicken granulosa cells express and actively secrete apolipoprotein A-I (apoA-I) as a part of particles with very high density. The granulosa cell-derived, apoA-I-containing material is different from the small portion of yolk high density lipoprotein that arises via transfer from the peripheral circulation. We propose that the ApoA-I-containing particles secreted by granulosa cells 1) support the growth of the rapidly growing germ cell, possibly by direct lipid transfer to the plasma membrane of the oocyte, and/or 2) deliver cholesteryl esters to the steroid-producing cells of the theca layer. These findings are discussed with respect to the proposed functions of apoE (an apolipoprotein not found in chicken) within the mammalian follicle.—Hermann, M., Lindstedt, K. A., Foisner, R., Mörwald, S., Mahon, M. G., Wandl, R., Schneider, W. J., Nimpf, J. Apolipoprotein A-I production by chicken granulosa cells. FASEB J. 12, 897–903 (1998)

Key Words: follicle • vitellogenesis • oocyte

	INTRODUCTION

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IN EGG-LAYING SPECIES, lipoprotein transport is a central element in sustaining a continuous flow of nutrients to the developing oocytes. In laying hens of the chicken (Gallus gallus), the best-studied model in this respect, a single oocyte takes up about 5 g of lipid and protein during the last 7 days of development. Most, if not all, of the yolk precursors are synthesized by the liver, secreted into the circulation, and taken up by the growing oocytes via receptor-mediated endocytosis (1). The major plasma-derived yolk precursors are the lipoproteins, very low density lipoprotein (VLDL),² and vitellogenin (VTG). The specific and efficient uptake of these two plasma-borne yolk precursors is mediated by a germ cell-specific splice variant of the avian homologue of the VLDL receptor (2–5). In contrast to VLDL and VTG, high density lipoprotein (HDL) particles enter the oocyte from the plasma independent of the receptor-mediated uptake pathway for VLDL and VTG (6). Besides the transport of these major yolk components, we have characterized transport of minor constituents that are also produced by the liver and taken up by the oocyte directly via the same receptor; these are riboflavin binding protein, which enters the oocyte proper complexed to VTG (7), and ₂-macroglobulin (8). Unlike the uptake of yolk material by specific receptors in the oocyte plasma membrane, little is known about the transport of these macromolecules through the different cell layers within the complex structure of the follicle (9). The oocyte is surrounded by the acellular perivitelline layer and by a monolayer of granulosa cells (GC), which are separated from the two-layered theca by a pronounced basement membrane. It is not known whether granulosa cells and/or the basement membrane affect the transport of the aforementioned high molecular weight yolk precursors from the capillaries of the theca to the plasma membrane of the oocyte. The current view is that this transport occurs via gaps between the GC (10).

Another interesting aspect is the contribution of GC to intercellular hormonal signaling in the follicle and to the production or transfer of molecules essential for rapid oocyte growth. Although parts of the perivitelline layer (11) and fibronectin, a major component of the basement membrane (12), are synthesized and secreted by GC, it is not clear whether GC-derived components are directly involved in oocyte development. In some invertebrates such as Drosophila melanogaster, major yolk components (e.g., vitellogenin) are not only produced extraovarially in the fat body, but also by nurse cells within the ovary. Thus, we have begun to examine the contribution of the GC layer to the production of oocyte components in vertebrate follicles.

	EXPERIMENTAL PROCEDURES

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Experimental animals and antibodies
Brown Derco laying hens (30–40 wk old) were obtained from Heindl Co. (Vienna, Austria) and maintained on layer's mash with free access to water and feed under a daily light period of 16 h. Polyclonal antibodies against chicken apolipoprotein A-I (apoA-I) were described previously (6).

Gel electrophoresis, Western blotting, and metabolic labeling
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4.5–18%), electrophoretic transfer of the proteins to nitrocellulose, and Western blotting were performed as described earlier (6).

For metabolic labeling, newborn chick skin and chicken follicles from which the vascularized theca layer had been mechanically removed were processed and incubated with [³⁵S]methionine according to ref 13. For immunoprecipitation, 200 µl of the incubation medium or cell extract was mixed with 10 µg of anti-apoA-I immunoglobulin G (IgG) or preimmune IgG and incubated for 4 h at 4°C. Twenty-five microliters of a 50% (w/v) suspension of protein-A Sepharose in phosphate-buffered saline (PBS) was added and the incubation was continued for another 12 h. The Sepharose was washed four times in 200 µl PBS containing 1% Triton X-100, resuspended in 40 µl 1x loading buffer, heated for 5 min at 95°C, and centrifuged for 1 min.

Northern blot analysis
Total RNA and poly A⁺-RNA prepared from GC sheets, cultured GC, and small vitellogenic follicles from laying hens were separated on 1.25% agarose gels (glyoxal) and blotted onto Hybond N⁺ nylon membrane (14). Hybridization with the full-length chicken apoA-I cDNA was performed at 65°C for apoA-I in 0.5 M sodium phosphate buffer (pH 6.8), 10 mg/ml bovine serum albumin (BSA), 70 mg/ml SDS, and 1 mM EDTA. Washing was performed at 65°C in 40 mM sodium phosphate buffer (pH 6.8), 5 mg/ml BSA, 50 mg/ml SDS, 1 mM EDTA and in 40 mM sodium phosphate buffer (pH 6.8), 10 mg/ml SDS, 1 mM EDTA. The filters were exposed to Reflection film (DuPont NEN, Wilmington, Del.) with intensifying screens at -80°C or Phospho Imager extension (Molecular Dynamics, Sunnyvale, Calif.) was performed for 4 days.

In situ hybridization
Follicles from the ovaries of adult chickens were processed for in situ hybridization exactly as described previously (2). RNA probes were prepared as follows. A 305 bp fragment (487–791) prepared from the chicken apoA-I cDNA by polymerase chain reaction amplification using a sense (5'-GCTCAGGAGCTGAAGGAGCTCACC) and an antisense (5'-TTCTCAGCATAGGGGGTGAGGCGC) primer was subcloned into the pGEM-T vector (Promega, Madison, Wis.). The purified plasmid was linearized and the RNA probe was prepared and labeled with digoxigenin-UTP by in vitro transcription, using an SP6 and T7 RNA polymerase [DIG RNA labeling Kit (SP6/T7)] according to the manufacturer's recommendation (Boehringer-Mannheim). The slides were mounted in Aquamount (BDH, Poole) and photographs were taken with a Zeiss Axiovert 10 light microscope.

Preparation of GC sheets and short-term culture of GC
Granulosa cell layers (GC sheets) were prepared from preovulatory follicles (F1–F5) as described (15). GC sheets were maintained in short-term culture in M199 medium supplemented with 10% lipoprotein deficient serum penicillin (50 U/ml), streptomycin (50 µg/ml), and L-glutamine (0.1 mg/ml) for at least 48 h without significant detachment of the cells. For isolation of granulosa cells, GC sheets were digested for 30 min at 37°C in 2 ml of M199 containing 4 mg collagenase and 0.15 mg trypsin inhibitor, with continuous gentle shaking. Finally, the tissue was mechanically disrupted with a Pasteur pipette and the GC were sedimented at 600 x g for 10 min. The cell pellet was washed with M199 and suspended (1 x 10⁶ cells/ml) in 3 ml M199 supplemented with 10% LPDS, penicillin (50 U/ml), streptomycin (50 µg/ml), and L-glutamine (0.1 mg/ml) in a 6 cm dish and incubated for 48 h at 37°C in 5% CO₂. Harvesting and Triton X-100 extraction of the cells were performed as described (16).

Separation of lipoproteins from cell media and chicken plasma
Lipoproteins in the incubation media of chicken follicles, in the culture media of granulosa cells, and in rooster plasma were separated by a step gradient ultracentrifugation according to ref 17. After centrifugation, 0.5 ml fractions were collected from the bottom of the tube, the density of each fraction was measured, and the apoA-I distribution was analyzed by Western blotting.

Immunofluorescence
Granulosa cell sheets were isolated from F2 or F3 follicles, attached to adhesion slides (BioRad, Hercules, Calif.), and fixed in methanol:aceton (1:1) for 5 min at -20°C. Samples were then blocked in PBS containing 0.2% gelatine for 30 min at room temperature and incubated with the respective antiserum diluted (1:100 in PBS/gelatine) for 60–120 min at room temperature. After several washes in PBS, samples were incubated with goat anti-rabbit conjugated to BODIPY FL (Molecular Probes, Leiden, Netherlands) for 60 min at room temperature. After several washes, slides were incubated in PBS containing 0.1 µg/ml propidium iodide for 10 min, washed in distilled water, and embedded in Mowiol (Hoechst, Frankfurt, Germany). Samples were viewed in a Zeiss Axiophot microscope and the MRC 600 laser scanning microscope (BioRad) equipped with a Krypton laser and a double filter set.

	RESULTS

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To study the production of potential oocyte components by granulosa cells, we established a short-term culture system for GC layers containing an intact basement membrane and perivitelline membrane. Immunoblotting of culture medium ( Fig. 1, lane 5) of a GC sheet revealed that apoA-I is present after 24 h incubation in apoA-I-free medium. We have recently shown that apoA-I is an integral component of HDL in yolk (6); thus, to exclude contamination of the medium with residual yolk as the source of apoA-I, we used as control primary GC cultured according to an established procedure involving trypsin treatment of the `sheet' (15). Granulosa cells cultured in this way also synthesize apoA-I. In addition to secreting large amounts of this protein into the medium (lane 4), the cells also contain detergent-extractable apoA-I ( Fig. 1, lane 3). Finally, large previtellogenic (diameter 3–4 mm) and small vitellogenic (diameter 5–6 mm) follicles kept in a short-term culture system secreted apoA-I into the medium (data not shown).

View larger version (107K): [in this window] [in a new window]   Figure 1. Detection of immunoreactive apoA-I in the media of cultured granulosa cells and granulosa cell sheets. Granulosa cell sheets were maintained in short-term organ culture and GC were grown in culture medium for 48 h. The cells were collected and extracted with Triton X-100 in a total volume of 150 µl, as described in Experimental Procedures. 10 µl of the respective media and of the GC extract were separated on a 12% SDS-polyacrylamide gel under reducing conditions, transferred onto nitrocellulose, and processed for immunoblotting using anti-apoA-I IgG. Lane 1: M199 medium; lane 2: M199 + lipoprotein-deficient serum; lane 3: Triton X-100 extract of cultured GC; lane 4: medium from cultured GC; lane 5: medium from cultured GC sheets. Exposure time was 10 s.

These data clearly demonstrate that GC secrete apoA-I, but do not prove that apoA-I is synthesized by these cells. It could well be that apoA-I containing HDL is transported through GC from the circulation at the thecal side to the oocyte via trans-cytosis. Thus, apoA-I detected in our experiments could have been released slowly from intracellular pools during the in vitro culture. To test whether GC actually synthesize apoA-I, we metabolically labeled small vitellogenic follicles (diameter, 5–6 mm) in vitro with [³⁵S]methionine. As demonstrated in Fig. 2, significant amounts of labeled apoA-I were immunoprecipitated from the incubation medium (lane 4) and, to a much lesser extent, from the high-speed supernatant of the follicles (lane 6). Parallel experiments with isolated GC sheets instead of total follicles gave similar results (data not shown). Newborn chick skin, previously shown to synthesize and secrete apoA-I (13), was used as control (lanes 7, 8).

View larger version (54K): [in this window] [in a new window]   Figure 2. Biosynthesis and secretion of apoA-I by chicken follicles. Small vitellogenic follicles (diameter, 4–5 mm) or slices of chicken skin were incubated in methionine-free medium in the presence of 0.25 mCi [35S]methionine as described in Experimental Procedures. The high-speed supernatant from follicle extracts (lane 2) and the cell-free medium of the follicle incubation (lane 3) were subjected to electrophoresis on a 4.5–18% SDS-polyacrylamide gel. Immunoprecipitations of proteins present in the cell-free medium of the follicle incubation were performed with anti-apoA-I IgG (lane 4) or an unrelated control IgG (lane 5). Immunoprecipitates of total follicular protein (lane 6), skin incubation medium (lane 7), and total skin protein (lane 8), using anti-apoA-I IgG, are shown. Lane 1, 14C-labeled methylated protein standards (BRL; lysozyme, 14.3 kDa; ß-lactoglobulin, 18.3 kDa; carbonic anhydrase, 29 kDa; ovalbumin, 43 kDa; BSA, 68 kDa; phosphorylase B, 97.4 kDa; myosin, 200 kDa). The gels were dried and exposed to X-ray film for 48 h at -70°C.

To test whether the GC layer is the sole site of apoA-I production in the follicle, we performed Northern blot analysis ( Fig. 3) and in situ hybridization experiments ( Fig. 4). Cultured GC ( Fig. 3, lane 1), GC sheets (lane 2), and small vitellogenic follicles (lane 3) express apoA-I mRNA. The difference in apoA-I signal intensities between the GC sheet and the total follicle might suggest that the oocyte within the follicle also expresses apoA-I. However, in situ hybridization of sections of follicles ( Fig. 4) clearly demonstrates that GC are the only cells within the follicle that express mRNA for apoA-I. The signal localized within lipid droplets in the yolk is artifactual, since these structures do not contain RNA. Thus, the difference observed in the expression levels of apoA-I in GC sheets and total follicle (cf. Fig. 3) most likely originates from the fact that GC sheets used in the in situ hybridization were prepared from F2 or F3 follicles, whereas follicular RNA was prepared from small (4–5 mm) follicles, a growth stage preceding F2 by at least 5 days. This experimental difference is unavoidable because 1) intact GC sheets cannot be obtained from small follicles, and 2) the yield of RNA from large vitellogenic follicles (F1–F4) is very poor due to the high yolk content. These results indicate that GC of small follicles express significantly more apoA-I mRNA than do GC of almost mature follicles. This might reflect the overall decrease in synthetic capacity of all follicular cells in the preovulatory phase and/or a reduced requirement for apoA-I in the late stages of oocyte growth.

View larger version (52K): [in this window] [in a new window]   Figure 3. Northern blot analysis of apoA-I transcripts in chicken follicular cells. Total RNA (25 µg/lane) isolated from cultured GC (lane 1), freshly prepared GC sheets (lane 2), and small vitellogenic (4–5 mm) follicles (lane 3) were denatured and separated by electrophoresis on a 1.25% agarose gel. Hybridization was performed with a 32P-labeled full-length chicken apoA-I cDNA and with a 32P-labeled 1.3 kb cDNA fragment of rat glyceraldehyde-3-phoshate dehydrogenase (GAPDH), as described in Experimental Procedures. Autoradiography was perfomed with intensifying screens for 24 h.

View larger version (114K): [in this window] [in a new window]   Figure 4. Localization of apoA-I transcripts in chicken follicles by in situ hybridization. Cryostat sections of small vitellogenic follicles (4–5 mm) were subjected to in situ hybridization with digoxigenin-labeled RNA probes (A, antisense; B, sense) corresponding to a 305 bp fragment (487–791) of chicken apoA-I cDNA. The probes were visualized as described in Experimental Procedures. The stained area in panel A represents the single-cell GC layer (g) surrounding the oocyte (o); t, theca layer. Bar, 25 µm.

Immunofluorescence studies using anti-apoA-I gave the typical staining pattern of a secreted protein ( Fig. 5A). The discrete punctate cellular staining gradually intensifies toward the cell surface, frequently outlining the cell borders of the GC, which are uniformly arranged in an epithelial-like pattern. The intense staining of the intercellular spaces might indicate that significant amounts of secreted apoA-I accumulate in the extracellular matrix before being passed on to theca cells or the oocyte. In contrast, receptor-associated protein, an endoplasmic reticulum resident protein that acts as a folding chaperone for members of the LDL receptor family (18) and is highly expressed in chicken GC (19), predominantly displays punctate staining in perinuclear regions and was not detectable at the cell surface and intercellular spaces ( Fig. 5C, D).

View larger version (105K): [in this window] [in a new window]   Figure 5. Immunofluorescent analysis of apoA-I in isolated granulosa cell sheets. Isolated GC sheets were attached to adhesion slides and processed for immunofluorescence microscopy using anti-ApoA-I antiserum (A) or anti-chicken receptor-associated protein (RAP) antiserum (C) in PBS/gelatine, followed by goat anti-rabbit IgG conjugated to BODYPY FL. To visualize nuclei, samples were double stained with with 0.1 µg/ml propidium iodide (B, D). Cells were viewed using the MRC 600 laser scanning microscope. Bar, 10 µm.

We subjected conditioned media from the ex vivo culture of GC sheets, cultured GC, or plasma from roosters to density gradient ultracentrifugation. Immunoblot analysis of gradient fractions ( Fig. 6) revealed that most of the apoA-I in plasma floats with a density of approximately 1.1 g/ml, characteristic of chicken HDL (20). Whereas less than 2% of the apoA-I in plasma floats at a density greater than 1.15 g/ml, all of the apoA-I in media obtained from GC cultures or GC sheets was found there. These results show that GC secrete apoA-I as a component of very high density particles, different from circulating HDL.

View larger version (37K): [in this window] [in a new window]   Figure 6. Density gradient analysis of apoA-I-containing particles secreted by granulosa cells. The incubation media from either the short-term culture of GC sheets or cultured GC and rooster plasma were subjected to density gradient ultracentrifugation as described in Experimental Procedures. The gradients were fractionated from the bottom (0.5 ml/fraction), and the densities of individual fractions were measured. The apoA-I content of the fractions was assessed by Western blotting according to Experimental Procedures.

	DISCUSSION

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Most of our knowledge about oocyte growth in avian species is related to the `liver–oocyte axis'. In contrast, very little is known about the contribution of GC to the growth of ovarian follicles. We have established an ex vivo GC layer organ culture and used it to examine the production of potential oocyte components by these cells. We demonstrate that GC produce and secrete apoA-I as a part of particles with very high density because 1) GC sheets and cultured GC secrete apoA-I-containing particles (<1.2 g/ml) into the culture medium; 2) intact follicles and GC sheets incorporate ³⁵S-methionine into secreted apoA-I; 3) immunohistochemistry shows apoA-I in the endoplasmic reticulum and secretory vesicles of GC and intercellular spaces; and 4) Northern blot analysis demonstrates the presence of apoA-I transcripts in the follicle and GC, and in situ hybridization confirms the localization of apoA-I message exclusively to the granulosa cells within the follicle.

Whereas chicken apoA-I is 49% identical to human apoA-I (21), the expression pattern of apoA-I in birds is significantly different from that in mammals. In chickens, apoA-I synthesis occurs in most tissues—notably the liver, intestine, kidney, ovary, muscle, brain, and skin (21)—whereas the expression of apoA-I in mammals is restricted to liver and intestine (22). However, the expression pattern of chicken apoA-I is very similar to that of mammalian apoE (23). Chicken apoA-I production increases at sites of peripheral nerve injury and regeneration, and this is true for mammalian apoE (24). Another common site of expression of apoE in mammals and apoA-I in chicken is the skin (13, 25). In this case, it was speculated that the apolipoproteins might serve a local lipid transport to build up the lipid-rich barrier of the stratum corneum. These observations, together with the fact that birds apparently do not produce apoE (20), have led to the hypothesis that chicken apoA-I can serve as a functional analog of mammalian apoE (13, 21).

This notion seems to be supported further by the fact that a short stretch of chicken apoA-I (residues 170–187) is particularly similar to the receptor binding domain of apoE (residues 132–149) (21). However, this speculation is not supported experimentally, e.g., by demonstrating that chicken apoA-I could bind to mammalian E-receptors (LDL receptor or LRP). Our own studies have demonstrated that chicken apoA-I is not a ligand for any of these receptors in the chicken (16, 26, 27). Together, these data argue against the hypothesis that apoA-I in the chicken is a functional counterpart of mammalian apoE, at least as it relates to metabolic pathways involving members of the LDL receptor gene family. Of course we cannot exclude the presence of a hitherto unidentified apoA-I receptor in birds that could bind and internalize apoA-I-containing particles; another possibility is that there are receptor-independent mechanisms of lipid transport or mobilization requiring apoA-I in birds or apoE in mammals.

Nevertheless, our data on apoA-I production in chicken GC do seem to point to a close relationship to apoE. In response to changes in intracellular cAMP levels, rat ovarian GC synthesize and secrete significant amounts of apoE (28). Although the function of this apoE is not established, progesterone production by ovarian GC in vitro has been shown to depend on the presence of lipoproteins in the medium (29). Rat GC express LDL receptors (30) and VLDL receptor transcripts are present in total rat ovarian tissue (31), but it is not yet known whether mammalian ovarian cells express LRP. In such a setting, apoE could either mediate the local transport of GC-derived lipids to thecal cells or it could be the substrate of a secretion-recapture pathway, as originally proposed for the liver (32). Alternatively, the production of apoE and receptor-mediated lipid transport between ovarian cells may be two independent processes. It was demonstrated that the spectrum of steroid hormones produced by theca cells depends on the presence of apoE in the medium (33). This points to a possible paracrine function of apoE in the mammalian ovary.

The current data, together with previous findings described above, challenge the proposed functional equivalence of mammalian apoE and chicken apoA-I with respect to receptor-mediated metabolsim. However, it is conceivable that in the chicken follicle, SR-BI, which recently was identified in mammals as a receptor for HDL (34, 35), might participate in local lipid transport from GC either to theca cells or to the growing oocytes. Such transport could provide cholesterol for steroid hormone synthesis in theca cells; alternatively (or in addition), the lipid component (or components) could be used by the rapidly growing oocyte for plasma membrane synthesis. This could be especially important for egg-laying species, since apparently neither cholesterol nor phospholipids from yolk precursors are used for this purpose (6). The unique lipoprotein metabolism in the laying hen, where massive amounts of VLDL are produced in the liver in order to sustain the development of the egg, might necessitate efficient local production of apoA-I-containing particles within the follicle. The production of apoE in the mammalian follicle might be unrelated to this apoA-I-based lipid transport, but could serve specific functions within the mammalian follicle, e.g., as a regulator of theca cell proliferation.

	ACKNOWLEDGMENTS

 
We appreciate the expert technical assistance by Romana Kukina and Harald Rumpler. This work was supported by grants from the Austrian Science Foundation (F-0603 to R.F., F-0608 to W.J.S., and F-0606 to J.N.). M.G.M. is supported by an Australian Postgraduate Research Award.

	FOOTNOTES

 
¹ Correspondence: Department of Molecular Genetics, Biocenter and University of Vienna, Dr. Bohrgasse 9/2, A-1030 Vienna, Austria. E-mail: JNIMPF{at}mol.univie.ac.at

² Abbreviations: apo, apolipoprotein; GC, granulosa cells; (V)LDL, (very) low density lipoprotein; VTG, vitellogenin; HDL, high density lipoprotein(s); LR8, LDL receptor relative with 8 ligand binding repeats; LRP, LDL receptor-related protein; Ig, immunoglobulin; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IgG, immunoglobulin G.

Received for publication June 27, 1997. Accepted for publication January 26, 1998.

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