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Biology of Reproduction 64, 1080-1089 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Apolipoprotein E Is a Putative Autocrine Regulator of the Rat Ovarian Theca Cell Compartment

Celina V. Zerbinattia, Loretta P. Mayera, Robert G. Audeta, and Cheryl A. Dyer1,a

a Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-5640

ABSTRACT

Apolipoprotein (apo) E inhibits androgen production by ovarian theca cells. We found that apo E, as a synthetic peptide mimicked the full-size protein, induced theca and interstitial cell (TIC) apoptosis indicated by pyknotic cell morphology, increased DNA end-labeling (TUNEL), and DNA ladders. None of the low-density lipoprotein (LDL) receptor superfamily members were involved because the universal antagonist of these receptors, receptor-associated protein (RAP), did not block apo E-induced apoptosis. Furthermore, several apo E synthetic peptides that do not bind the LDL receptor did induce TIC apoptosis. Similar to apo E, apoptogenic agents such as ceramide and LY 294002, a phosphatidylinositol (PI) 3-kinase inhibitor, induced apoptosis and suppressed androstenedione production. However, apoptosis alone was not responsible for apo E suppression of androstenedione production because both insulin and IGF-I prevented apo E-induced apoptosis, but neither restored androstenedione production. Theca cells of atretic follicles express the greatest apo E mRNA, and here we show that cultured TIC produce apo E. When considered with the observation of TUNEL-positive theca cells in atretic follicles these results support our hypothesis that intraovarian apo E controls theca cell production of androgen as well as limiting the size of the theca cell compartment.

apoptosis, insulin, theca cells

INTRODUCTION

Apoptosis is a carefully orchestrated means by which cells that have served their purpose are eliminated from the ovary. Apoptosis in ovarian granulosa and luteal cells has been the subject of intensive investigation [1]. Far less studied is the regulation of apoptosis in the theca interna cell compartment of the ovary. In contrast to the granulosa cell population, which is entirely eliminated via apoptosis, not all theca cells of atretic follicles become apoptotic [2–5]. Theca cells that are not eliminated by apoptosis transition into secondary interstitial cells whose steroidogenic phenotype is to produce progesterone rather than androgen, which is the major steroid product of theca cells [6].

To date the only reported initiators of theca cell apoptosis in vitro are transforming growth factors (TGF){alpha} + ß [7, 8]. Also, theca cell apoptosis is mediated by the Fas ligand/receptor system involving the sphingomyelin pathway that uses ceramide as an intermediate signaling molecule [9]. Because TGF{alpha} and TGFß are expressed by theca of atretic follicles, it has been suggested that these growth factors are autocrine regulators that maintain ovarian homeostasis by eliminating theca cells [10]. Additional mediators of theca cell apoptosis have yet to be identified and characterized.

We have been investigating the actions of apolipoprotein (apo) E on ovarian theca and interstitial cells (TIC). Although apo E is most often considered in the context of cholesterol transport in the plasma [11], numerous reports have described local production of apo E in steroidogenic tissues, particularly in the ovary [12, 13]. The TIC express significant levels of apo E mRNA and, importantly, the theca cells of atretic follicles express the greatest amounts [13]. Exogenous apo E exerts a selective and concentration-dependent effect on theca cell androgen production in vitro [14]. At lower concentrations, apo E stimulates theca cell androgen production via its interaction with one or more members of the low-density lipoprotein (LDL) receptor superfamily [15]. In contrast, at higher concentrations, apo E selectively inhibits androgen production without suppressing the production of progesterone [14, 15]. Because TIC treated with higher concentrations of apo E appeared shrunken and pyknotic, we suspected that the cells were undergoing apoptosis. Here we examined TIC production of apo E, apo E's ability to induce apoptosis in TIC via members of the LDL receptor superfamily, and analyzed the capability of anti-apoptotic insulin and insulin-like growth factor-I (IGF-I) to prevent apo E's inhibitory activities.

MATERIALS AND METHODS

Cell Preparation

Whole ovarian dispersates containing theca cells were obtained from immature Sprague-Dawley rats that had been hypophysectomized at 21 days of age [14]. The dispersed cells were enriched for TIC content by Percoll density gradient separation, and >90% are positive for 3ß-hydroxysteroid dehydrogenase activity [16]. Only theca cells produce androgens in this culture setting [17]. Animal care was in accordance with the Institutional Animal Care and Use Committee of Northern Arizona University. The cells were plated at 30 000–50 000 cells per well of a 96-well plate (Costar, Cambridge, MA) in 0.25 ml of serum-free McCoys 5a modified medium with penicillin and streptomycin (Life Technologies, Grand Island, NY) and incubated in a 95% air, 5% CO2, humidified incubator at 37°C. The next day, LH (NIDDK-oLH-25; 2.3 U/mg, 1 U = activity of 1 mg of NIH-LH-S1) and the other agents were added. In all experiments, except where noted, a concentration of 1 ng/ml of LH was used because it induces maximal stimulation of theca cell androstenedione production [17]. N-Acetyl-D-erythro-sphingosine (C2 ceramide) was obtained from CalBiochem (San Diego, CA), dimethyl sulfoxide (DMSO) and insulin were obtained from Sigma Chemical Company (St. Louis, MO), LY294002 was obtained from Alexis (San Diego, CA), and recombinant human IGF-I was obtained from Biosource International (Camarillo, CA). The apo E synthetic peptide was kindly provided by Dr. L.K. Curtiss of The Scripps Research Institute, La Jolla, CA, and receptor-associated protein (RAP) was kindly provided by Dr. D. Strickland of the American Red Cross, Bethesda, MD.

Ovarian Sections Assayed by TUNEL

Ovaries obtained from hypophysectomized rats were fixed in Bouin's fixative for 1 h followed by paraffin embedding. The presence of apoptotic nuclei in the ovarian sections (6 µm) was detected using a TUNEL kit from Promega (Madison, WI) according to the manufacturer's protocol with the following modifications. After deparaffinizing and rehydration, the endogenous peroxidase activity was not quenched but the sections were microwaved in Tris buffer (pH 10.6) for a total of 10 min (3 min at 750 W and 7 min at 450 W) at 99°C to increase TUNEL labeling. The slides were examined using an Aristoplan Leitz microscope with PL Fluotar 25x and a PL Apo 100x oil immersion lenses. Images were obtained with a Spot-cooled color digital (charge-coupled device) camera and software (Spot, version 1.1.02 for Windows 3.1; Diagnostics Instruments, Sterling Heights, MI).

Apolipoprotein and Synthetic Peptide Preparations

Both recombinant human apo E3 from PanVera Corporation (Madison, WI) and purified human apo A-I from Biodesign International (Kennebunk, ME) were exhaustively dialyzed against PBS before their addition to the cells. The apo E peptide used for all of the studies was synthesized and purified as previously described [15]. The preparation and characterization of the modified apo E peptides and the RAP peptide is described in detail in the study of their LDL receptor binding activity [18]. The purity of the synthetic peptide preparations ranged from 93% to 100% and averaged 60%–70% peptide content. The parent apo E peptide sequence, (141–155)2, to which all other sequences were compared for activity, was chemically modified by 1,2-cyclohexanedione treatment that introduces a negative charge on arginine residues or by acetic anhydride treatment that introduces a negative charge on lysine residues [19, 20]. All peptides were dialyzed exhaustively against PBS in 1000 molecular weight cut-off tubing before their addition to the cells.

Analysis of Steroid Production

After 48 h of culture the TIC supernatants were collected and stored at -20°C until the content of progesterone and androstenedione were measured in specific RIAs [14]. The results of the RIAs were calculated by four-parameter logistic analysis using the software AssayZap (BioSoft, Ferguson, MO). Each data point was done in quadruplicate and the results are the mean ± SEM. The results marked with an asterisk were statistically different at a significance of P < 0.05 as determined by Student t-test or one-way ANOVA with pairwise multiple comparison of the mean responses among the different treatment groups by Student-Newman-Keuls test using the software SigmaStat version 2.03 (SPSS Inc., Chicago, IL).

Western Blot Analysis of TIC Apo E Production

The TIC (40 000 cells per well per 0.25 ml medium) were cultured with increasing LH (0, 1.0, 3.0, 10.0 ng/ml) ± human high-density lipoprotein (HDL; 100 µg protein/ml). The human HDL was generously provided by Dr. C.L. Banka of The Scripps Research Institute, La Jolla, CA [21]. After 1 wk of culture the supernatants were collected and pooled from eight replicate wells, concentrated approximately 20-fold, and an equivalent volume of each sample run on a 4%–20% gradient SDS-PAGE. The transferred proteins were identified using a mouse monoclonal antibody to rat apo E that was detected using the Rad-Free chemiluminescence kit from Schleicher & Schuell (Keene, NH), according to the manufacturer's protocol.

Light Microscopy and TUNEL of TIC

For light microscopy, TIC were cultured in chambered glass slides with the same dimensions and media volume that were used for cells cultured in the 96-well plate format. After a 24-h incubation, the culture supernatants were collected for analysis of steroid content, and the cells were fixed and processed according to the procedure of Kerr et al. [22]. Sections (0.5–1.0 µm) were examined in an Aristoplan Leitz microscope (Vermont Optechs, Charlotte, VT) with a PL Apo 63x oil immersion lens. The presence of DNA fragments in TIC was detected using a fluorescence-based TUNEL kit from Promega (Madison, WI) according to the manufacturer's protocol.

DNA Isolation and Fragment Analysis

The DNA ladders were detected according to the method of Tilly and Hsueh [23]. In brief, low molecular weight DNA was extracted from TIC pooled from six to eight wells and analyzed on 2.5% NuSieve agarose gel from FMC BioProducts (Rockland, ME). The presence of DNA was detected with SYBR Gold fluorescent dye from Molecular Probes (Eugene, OR), and the image was analyzed using Kodak Digital Science Image analysis software (Rochester, NY). To measure the extent of DNA fragmentation, 100 ng of DNA from each sample was labeled at 3'-ends with [{alpha}-32P]dideoxy-ATP (specific activity ~ 3000 Ci/mmol) Amersham Pharmacia Biotech (Piscataway, NJ) using TdT from Promega. Labeled DNA (50 ng/ml) was separated on a 1.8% agarose gel, and after autoradiography the portions of each lane corresponding to DNA <10 kilobases were cut and counted in a ß-scintillation counter.

Cell Proliferation Assay

The TIC number was measured using the Quantos cell proliferation assay from Stratagene Cloning Systems (La Jolla, CA) according to the manufacturer's protocol. The fluorescent signal was measured in a plate reader and calibrated to the standard curve that was generated from flash-frozen, freshly isolated TIC.

RESULTS

Theca Cells of Atretic Follicles Were TUNEL-Positive

In atretic follicles most attention has been directed to detecting the presence of apoptotic granulosa cells. We examined paraffin-embedded ovarian sections for the coincidence of follicular atresia and apoptotic theca cells. As seen in Figure 1, A and B, both granulosa cells (G) and theca cells (T) of this atretic follicle were TUNEL-positive, indicating apoptosis.



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  		FIG. 1. Atretic follicles have TUNEL-positive theca cells. Apoptotic cells were detected by TUNEL in paraffin-embedded ovarian sections. A) Low magnification (x25) image of an atretic preantral follicle identified by numerous TUNEL-positive cells (arrowheads) in the granulosa cell compartment (G), and the theca cell layer (T) also contained a few TUNEL-positive cells indicated by the }. B) The higher magnification (x100) image of TUNEL-positive theca cells shown in A is indicated by arrows. Data shown are representative of five ovaries

Cultured TIC Produce Apo E

Instances of cellular apo E mRNA levels not paralleling the level of apo E protein production [24] prompted us to examine TIC apo E production in vitro. The TIC were cultured in serum-free medium for a week with increasing LH concentrations ± human HDL at 100 µg/ml. As shown in Figure 2, apo E was not detected in supernatants from TIC that had been cultured without HDL (lanes 1–4) but was produced in a dose-response manner to increasing LH concentrations when human HDL was present (lanes 5–8). Even in the absence of LH stimulation, apo E production was detected with only HDL present (lane 5).



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  		FIG. 2. Cultured TIC produce apo E only when human HDL was present. The TIC (40 000 cells/well) were cultured with increasing LH concentrations ± human HDL for 1 wk. Pooled supernatants from eight replicate wells were concentrated, and the apo E content was detected by Western blot analysis. The left lane contains the molecular weight standards with the 30 000 molecular weight band identified. Lanes 1–4 are the samples from TIC treated with LH concentrations of 0, 1.0, 3.0, 10.0 ng/ml; lanes 5–8 are samples with the same LH concentrations + human HDL (100 µg protein/ml). The far right lane is 20 µg of rat serum. Data shown are representative of two experiments

Apoprotein E Peptide Induced Apoptosis in TIC

Exogenous apo E, added either in the full-size protein or as a synthetic peptide mimic selectively suppresses TIC androgen production without altering the production of progesterone [14, 15, 17]. At apo E concentrations >=0.5 µM, we observed that the cells were pyknotic and an abundance of apoptotic bodies. Shown in Figure 3A, left side, is a cluster of only LH-treated TIC that have dispersed nuclear heterochromatin. Shown in Figure 3A, right side, is a cluster of TIC treated with LH + 1.0 µM apo E peptide with abundant fragmented cell debris, apoptotic bodies, and condensed chromatin—all are morphological features of apoptosis. Also shown in Figure 3B is TUNEL staining (green fluorescence) that indicated increased DNA fragmentation occurred with increasing apo E peptide concentrations from 0.5 to 2.0 µM.



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  		FIG. 3. Apoprotein E induced TIC apoptosis. A) The TIC were stimulated with LH ± apo E peptide (1.0 µM) for 24 h when the cells were fixed and processed for light microscopy. Final magnification x63. B) The TIC were stimulated with LH in the presence of increasing concentrations of the apo E peptide (0.5, 1.0, 2.0 µM). After 48 h of culture the cells were fixed and stained with propidium iodide (red fluorescence), and DNA fragmentation was detected using TUNEL (green fluorescence). Final magnification x40. Data shown are representative of two experiments

Another hallmark of apoptosis is the presence of 180- to 200-base pair multimers of DNA or DNA ladders. As shown in Figure 4, there was an increase in DNA ladders in TIC that were cultured with apo E peptide concentrations of 0.5 and 1.0 µM (lanes 2 and 3, respectively) when compared to cells treated with only LH (lane 1).



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  		FIG. 4. The apo E peptide induced the formation of DNA ladders independent of the LDL receptor superfamily. The TIC were cultured for 48 h with LH (lane 1), LH + apo E peptide 0.5 µM (lane 2), LH + apo E peptide 1.0 µM (lane 3), RAP 0.5 µM pretreatment for 15 min + LH (lane 4), RAP 0.5 µM pretreatment for 15 min + LH + apo E peptide 1.0 µM (lane 5). The low molecular weight DNA was extracted from the cells and separated on an agarose gel. The DNA molecular weight markers are on the far right side of the gel. Data shown are representative of two experiments

Members of the LDL Receptor Superfamily Were Not Involved in Apo E-Induced TIC Apoptosis

The RAP is a universal antagonist that prevents apo E binding to the members of the LDL receptor superfamily and because of this property is used to test if actions of apo E are due to its interaction with one or more of these receptors [25, 26]. As we have previously reported, the apo E-mediated inhibition of androstenedione production is not prevented by RAP [15, 17]. Consistent with this finding, the DNA ladders induced by apo E peptide at 1.0 µM (Fig. 4, lane 3) were not blocked by RAP (0.5 µM) pretreatment of the cells (Fig. 4, lane 5). The DNA ladders in cells treated with LH + RAP (0.5 µM) but without the apo E peptide present (Fig. 4, lane 4) were similar to DNA ladders in cells treated with only LH (Fig. 4, lane 1).

Additional evidence that the members of the LDL receptor gene family were not involved in apo E inhibitory activities was provided by analysis of the activities of 11 deleted, substituted, or chemically modified apo E synthetic peptides. These peptides were derived from amino acids that are residues 141–155 of apo E, LRKLRKRLLRDADDL, repeated in a linear tandem or dimer (141–155)2 peptide that binds both the LDL receptor [18] and the LDL receptor-related protein or LRP [27]. Of the 11 peptides, 3 peptides had single amino acid substitutions, 3 peptides had amino acid residues deleted, 2 peptides had the lysine or arginine residues chemically modified, and 2 peptides were single repeats of the 141–155 amino acids—one within the longer sequence of 129–162 amino acids of apo E, and the other a single repeat of 141–155 or the monomer peptide. Increasing concentrations of these peptides were added to TIC cultures, and androstenedione and progesterone production were measured after 48 h. The effective inhibitory concentrations that caused 50% inhibition (IC50) of androstenedione production were calculated using the four parameter logistic analysis of each curve (Table 1). The peptides are ranked in Table 1 by decreasing potency. Also in Table 1, from a previous study, are the concentrations of each peptide that inhibit LDL binding to its receptor on human skin fibroblasts [18]. Ten of the 11 apo E peptides tested inhibited androstenedione production. Only the deletion peptide, (145–155)2, did not inhibit androstenedione production. Of the 10 peptides that inhibited androstenedione production, only 5 compete with LDL binding to its receptor and are identified in Table 1 by an asterisk. Progesterone production by the apo E peptide-treated TIC was unchanged (data not shown). All apo E peptides that inhibited androstenedione production induced apoptosis (data not shown) that was indicated by distinct morphological changes similar to those seen in Figure 3A. For comparison to the apo E peptides, we have included the IC50 concentration of 1.8 µM for recombinant human apo E3 (Table 1) [15]. To demonstrate that apo E inhibition of androstenedione production and induction of TIC apoptosis was dependent on an apo E-specific amino acid sequence, we tested the activity of a RAP synthetic peptide. This peptide is homologous to apo E in its enrichment for lysine and arginine residues but does not have them in the same sequence as in apo E [28]. The RAP peptide did not inhibit androstenedione production, induce TIC apoptosis, or compete with LDL binding to its receptor (Table 1).


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  		TABLE 1. Biological activities of modified apo E peptides, RAP peptide, and human apo E3.

Induction of TIC Apoptosis Was Specific to Apo E

We have shown that recombinant human apo E3 selectively inhibits androstenedione production by TIC [15]. To confirm that native apo E also induces TIC apoptosis, we compared the extent of DNA fragmentation caused by recombinant human apo E to that of the apo E peptide. Because the apo E peptide is more potent than recombinant human apo E in suppressing TIC androgen production [15] we tested the recombinant human apo E at a higher concentration than the apo E peptide. Isolated, lipid-free human apo A-I was also tested to confirm the specificity of apo E induction of TIC apoptosis. Only the apo E peptide at 1.0 µM (Fig. 5, lane 2) and human apo E3 at 2.0 µM (Fig. 5, lane 3) induced TIC apoptosis as indicated by more DNA ladders relative to cells treated with only LH (Fig. 5, lane 1). Apoprotein A-I at 2.0 µM, did not inhibit androstenedione production (data not shown) nor induce apoptosis in TIC (Fig. 5, lane 4).



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  		FIG. 5. Recombinant human apo E3 induced TIC apoptosis but apo A-I did not. The TIC were cultured for 48 h with LH (lane 1), LH + apo E peptide 1.0 µM (lane 2), LH + recombinant apo E3 2.0 µM (lane 3), and LH + apo A-I 2.0 µM (lane 4). The low molecular weight DNA was extracted from the cells and separated on an agarose gel. The DNA molecular weight markers are on the left side of the gel. Data shown are representative of two experiments

Apoptosis and the Selective Inhibition of TIC Androstenedione Production Were Coincident

In all cases in which apo E induced apoptosis, androstenedione production was suppressed to levels that were 50%–80% of control levels, while progesterone production was unaffected or sometimes increased (data not shown). The selective inhibition of androstenedione production may have resulted from apo E-mediated elimination of androgen-producing cells. To test this hypothesis we used ceramide, a defined inducer of TIC apoptosis [9]. Ceramide (10.0 µM) induced DNA ladder formation in TIC (Fig. 6A, lane 2) compared to control cells treated with only LH (Fig. 6A, lane 1). In these cells, androstenedione production was almost completely inhibited by ceramide treatment, but progesterone production was unchanged (Fig. 6B).



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  		FIG. 6. Ceramide induced TIC apoptosis and selectively inhibited androstenedione production. A) The TIC cells were cultured for 48 h with LH (lane 1) or LH + ceramide 10.0 µM (lane 2). The low molecular weight DNA was extracted from the cells and separated on an agarose gel. The DNA molecular weight markers are on the far right side of the gel. B) Supernatants from the above cultures were collected and the progesterone and androstenedione content measured by specific RIA. Each point is the mean ± SEM of four replicates and is representative of three experiments. *P < 0.05 by Student t-test

The inhibition of phosphatidylinositol (PI) 3-kinase activity has also been shown to induce apoptosis. We tested the possibility that a specific inhibitor of PI 3-kinase, LY294002, could induce apoptosis and inhibit androstenedione production in TIC. For this and the remaining assessments of apoptosis we used a quantitative assay of DNA fragmentation [23]. As shown in Figure 7A, increasing concentrations of LY 294002 selectively inhibited androstenedione production without altering progesterone production. The LY 294002 also induced DNA fragmentation as shown in Figure 7B. As with apo E, ceramide and LY 294002 treatments resulted in the coincidence of TIC apoptosis and the selective inhibition of androstenedione production.



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  		FIG. 7. Inhibition of PI 3-kinase induced TIC apoptosis and selectively inhibited LH-stimulated androstenedione production. A) The TIC were pretreated with LY 294002 (0–10.0 µM) for 1 h before the addition of LH. After 48 h of culture the supernatants were collected, and the progesterone and androstenedione content were measured by specific RIA. Each point is the mean ± SEM of four replicates and is representative of three experiments. B) The TIC were pretreated with LY 294002 (1.0–100.0 µM) for 1 h when LH was added, and the cells were cultured for 48 h. The low molecular weight DNA was end-labeled and separated on an agarose gel. The percentage of end-labeling relative to LH control cells was averaged from three experiments. *P < 0.05 by ANOVA

Insulin and IGF-I Prevented Apo E-Induced Apoptosis but Did Not Restore Androstenedione Production by TIC

Because LY294002-mediated inhibition of TIC PI 3-kinase selectively suppressed androgen production and induced apoptosis, and, given that insulin and IGF-I prevent apoptosis thru their activation of PI 3-kinase [29, 30], we tested insulin and IGF-I to see if they could attenuate apo E's actions. As shown in Figure 8, without apo E present, treatment with insulin or IGF-I stimulated androstenedione production but without an increase in progesterone production. These same doses of insulin (100.0 nM) and IGF-I (10.0 ng/ml) prevented apo E-induced apoptosis because DNA fragmentation was the same in cells treated with insulin or IGF-I as in cells treated with only LH (Fig. 9A). However, in the presence of apo E, only insulin was able to restore 20% of androstenedione production, while IGF-I treatment did not restore androstenedione production at all (Fig. 9B).



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  		FIG. 8. Insulin and IGF-I selectively stimulated TIC androstenedione production. The TIC were cultured with LH, LH + insulin (100.0 nM), or LH + IGF-I (10.0 ng/ml) for 48 h when the culture supernatants were collected. The progesterone and androstenedione content was measured by specific RIA. Each point is the mean ± SEM of four replicates and is representative of three experiments. *P < 0.05 by ANOVA



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  		FIG. 9. Insulin and IGF-I prevented apo E-induced apoptosis. The TIC were cultured for 48 h with LH + apo E peptide (1.0 µM), LH + apo E peptide + insulin (100 nM), or LH + apo E peptide + IGF-I (10.0 ng/ml). A) The low molecular weight DNA was end-labeled and separated on an agarose gel. The percentage of end-labeling in the presence of the apo E peptide relative to its respective control (LH, LH + insulin, or LH + IGF-I) was averaged from three experiments. B) Culture supernatants from the above treatments were collected, and androstenedione content was measured in a specific RIA. Each point is the mean ± SEM of four replicates and is representative of three experiments. *P < 0.05 by ANOVA

Because both insulin and IGF-I stimulate TIC proliferation [31–33] we determined if the anti-apoptotic effect of these treatments was due to a change in cell number. While insulin protected cells from apoptosis without inducing cell proliferation, IGF-I treatment at 10.0 ng/ml did increase cell number by 25% (Table 2).


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  		TABLE 2. The TIC number after culture with insulin and IGF-I

Activity of PI 3-Kinase Was Not Directly Involved in the Apo E Peptide Inhibitory Activity

Because insulin and IGF-I prevented apo E-induced TIC apoptosis, and their anti-apoptotic action in other cells is mediated by PI 3-kinase activation, we tested if the apo E peptide inhibitory activity was via suppression of PI 3-kinase activity. As shown in Figure 10A, the dose of LY 294002 that reduced androstenedione production by ~50% in LH-treated cells, potentiated the suppression of androstenedione production by cells treated with LH + insulin or LH + IGF-I. These results indicated that the stimulatory effect of insulin and IGF-I on TIC androgen production (Fig. 8) was probably mediated in part via PI 3-kinase activation. However, in the presence of the apo E peptide, LY294002 did not potentiate the inhibition of androstenedione production when compared to only LH-treated cells (Fig. 10B). These results indicated that PI 3-kinase was probably not involved in apo E peptide inhibition of androstenedione production and were consistent with the results shown in Figure 9B, where insulin and IGF-I were unable to prevent apo E peptide inhibition of androstenedione production even though they prevented apo E-induced TIC apoptosis (9A).



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  		FIG. 10. Inhibition of PI 3-kinase potentiated the action of insulin and IGF-I but did not potentiate apo E inhibition of androstenedione production. A) The TIC were pretreated with LY 294002 (1.0 µM) for 1 h before the addition of LH, LH + insulin, or LH + IGF-I. B) The LY 294002 (1.0 µM) was added for 1 h before the addition of LH + apo E peptide (0–1.0 µM). After 48 h of culture the supernatants were collected, and the androstenedione content was measured by specific RIA. Each point is the mean ± SEM of four replicates and is representative of three experiments. *P < 0.05 by ANOVA

DISCUSSION

Apoprotein E induced TIC apoptosis that was indicated by characteristic morphologic appearance of the cells, the presence of apoptotic bodies, and increased DNA fragmentation and DNA ladder formation. Both native apo E and the apo E peptide induce neuron apoptosis [34, 35]. The apo E peptide induces apoptotic-like cell death when present at concentrations >1.0 µM in neuronal cell cultures [35]. Apoprotein E peptide-mediated neuron apoptosis is preceded by DNA condensation and fragmentation but is not prevented by protein synthesis inhibitors, caspase inhibitors, or genetic deletion of bax [35]. Similar to these findings, we found that inhibitors of caspase 1 and 3 did not prevent apo E induction of apoptosis in TIC (unpublished data). A thorough characterization of apo E-induced TIC apoptosis is necessary to define the mediators of the pathway.

Apoprotein E induced TIC apoptosis at low micromolar concentrations that occur within the ovary. The best estimate of intraovarian apo E concentration is derived from analysis of human follicular fluid. The apo E concentration in the follicular fluid from cycling women varies from 0.5 to 5 times that of the plasma apo E concentration, which averages 1.2 µM in normolipidemic premenopausal women [36]. Thus, the apo E concentrations that induce apoptosis in TIC in vitro are well within the intraovarian physiologic range. In addition, as we have found before in comparing the biologic potencies of plasma-derived apo E to macrophage-derived apo E [37], it is very likely that apo E produced within the ovary is more potent than purified or recombinant apo E preparations.

A high level of apo E mRNA is expressed by theca cells of atretic follicles [13]. Here, we found that TIC apo E mRNA is translated and secreted as the mature protein. The TIC apo E secretion was only apparent when human HDL was present in the medium. Because increased apo E production is observed when cellular cholesterol content increases [38], we propose that HDL increases apo E production by raising TIC cholesterol content. High-density lipoprotein probably raises cellular cholesterol content by binding the scavenger receptor class B, type I that is expressed on TIC [39–41]. Consistent with findings in cholesterol-loaded macrophages [38], astrocytes [42], and adrenal cells [43], we predict that the increased TIC cholesterol content stimulates apo E production. In addition, an alteration in cellular lipid homeostasis caused by HDL may also inhibit apo E intracellular degradation [44], thereby increasing apo E production.

Apoprotein E-induced apoptosis did not depend on its binding to members of the LDL receptor superfamily. The RAP, which is a universal antagonist that prevents apo E binding to all members of the LDL receptor superfamily [25, 26], did not prevent apo E-induced apoptosis or the inhibition of androstenedione production. Similar to our findings in TIC, the apo E peptide has been shown to induce apoptosis in neuronal cells via RAP-insensitive pathways [35]. Additional evidence that the LDL receptor superfamily did not mediate apo E's inhibitory activity came from the results using apo E synthetic peptides that had been modified to ablate or attenuate LDL receptor binding activity [18]. We have previously demonstrated that apo E synthetic peptides containing a linear tandem repeat of the LDL receptor binding domain, amino acids 140–150, are able to bind the LDL receptor [18, 45]. However, apo E synthetic peptides with only one repeat of amino acids from 140 to 150 do not bind the LDL receptor [18]. We found that apo E peptides that do not bind the LDL receptor inhibited androstenedione production. For example, the proline substitution in the parent sequence of the apo E peptide (141–155)2 produces a peptide that has no measurable {alpha}-helical structure determined by circular dichroism and consequently does not bind the LDL receptor [18]. Nevertheless, this peptide was nearly as potent at inhibiting TIC androstenedione production as the parent apo E peptide sequence.

It appears that the structural requirement for apo E synthetic peptides to be inhibitory is a minimum number of positive charge amino acids in an active conformation. However, the specific spatial array of the lysine and arginine residues was not as important for apo E to induce apoptosis and inhibit androgen production as it is for the peptide to compete with LDL binding to its receptor [18]. Arginine residues were necessary for apo E inhibition of androgen production because when they were chemically modified to have a negative charge, the IC50 for the modified peptide was increased 10-fold. There was an apo E-specific amino acid sequence required to suppress androstenedione production and induce apoptosis. This is indicated by the RAP peptide that contains several lysine and arginine residues, similar to the apo E peptide sequence, but does not have the correct order or conformation for it to be active either in the TIC or to compete with LDL binding to its receptor. The requirement for correctly positioned, positive charge residues for apo E activity was also supported by the result that apo A-I did not alter TIC androgen production or induce apoptosis. The structure of apo A-I is similar to apo E, as they both have extensive amphipathic helix content, are highly lipophilic, and can microsolubilize cell membranes [46]. But apo E differs from apo A-I by its increased content of lysine and particularly arginine residues.

At first we thought that apo E's selective inhibition of androstenedione production resulted from apo E's selective elimination of androgen-producing theca cells, leaving the interstitial cells to produce progesterone. However, because the other apoptotic agents we tested, ceramide and LY 294002, mimicked the apo E selective inhibition of androstenedione production, it appears that the specific changes in steroid production that accompany apoptosis may be a general response. Progesterone production is maintained and even increased in granulosa cells undergoing apoptosis [47]. One explanation for this paradox is the increased proximity of the steroidogenic organelles that occurs with reorganization of the cytoskeleton during the initial steps of apoptosis favoring progesterone synthesis, which could also explain the sustained production of progesterone by apoptotic TIC that we observed.

Insulin and IGF-I treatments prevented apo E induction of apoptosis. In other cell types, insulin and IGF-I prevent apoptosis by activating PI 3-kinase as well as mitogen-activated protein kinase (MAPK) [29, 30]. Insulin-like growth factor-I was more effective than insulin in preventing apo E-induced apoptosis. The IGF-I is also more potent than insulin in preventing the spontaneous apoptosis of cultured preovulatory follicles [48]. The IGF-I inhibits stress-activated protein kinase/c-jun amino terminal kinase (SAPK/JNK) [49]. The SAPK/JNK phosphorylates transcription factors such as c-jun, and sustained phosphorylation of c-jun is associated with the apo E peptide-mediated apoptosis in neurons [35]. Another apo E mechanism of action is to reduce MAPK activity in smooth muscle cells [50]. Therefore, insulin and IGF-I may have prevented apo E induction of apoptosis by inhibiting the activation of SAPK/JNK and by activating MAPK. Insulin and IGF-I counteracted apoptosis induced by apo E, but they did not restore androstenedione production to control levels, suggesting that apo E acted independently of insulin or IGF-I signaling pathways to inhibit androstenedione production. This conclusion is also supported by the finding that inhibition of PI 3-kinase did not potentiate apo E inhibition of androstenedione production.

Theca cell-derived androgens are key in controlling the destiny of a follicle. Androgen is required for follicular development because its level controls how much estrogen is synthesized to stimulate follicular maturation. However, if too much androgen is made by the theca cells, the follicle becomes atretic as the granulosa cells are eliminated by androgen-mediated induction of apoptosis [51]. We propose that apo E regulates the theca cell compartment by controlling the expression of P450 17{alpha}-hydroxylase, C17–20 lyase, the rate-limiting synthetic enzyme for androgen production, and by eliminating androgen-producing theca cells by apoptosis. Therefore, apo E may exert an important regulatory role in follicular development and atresia.

ACKNOWLEDGMENTS

We thank Marilee Sellers for her technical assistance, Dr. L. Fritz for the use of his fluorescence microscope, and Dr. R. Markle for sharing his laboratory resources. We are grateful to Ms. Suzie Larson for her administrative assistance.

FOOTNOTES

First decision: 25 May 2000.

1 Correspondence: Cheryl A. Dyer, Department of Biological Sciences, Box 5640, South Beaver Street, Northern Arizona University, Flagstaff, AZ 86011-5640. FAX: 520 523 7500; cheryl.dyer{at}nau.edu Back

Accepted: November 10, 2000.

Received: April 27, 2000.

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