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Am J Physiol Endocrinol Metab 293: E1053-E1061, 2007. First published August 14, 2007; doi:10.1152/ajpendo.00554.2006
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Lipoprotein receptor expression during luteinization of the ovarian follicle

Leonor Miranda-Jiménez1,2 and Bruce D. Murphy1

1Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada; and 2Campus San Luis, Colegio de Postgraduados, Salinas, San Luis Potosi, Mexico

Submitted 11 October 2006 ; accepted in final form 11 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ovarian follicles luteinize after ovulation, requiring structural and molecular remodeling along with exponential increases in steroidogenesis. Cholesterol substrates for luteal steroidogenesis are imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating high-density lipoproteins and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). An 82-kDa form of SR-BI predominates throughout, is weakly present in granulosa cells, and is robustly expressed in the CL, along with the less abundant 57-kDa form. Digestion of N-linked carbohydrates substantially reduced the SR-BI mass in luteal cells, indicating that differences between forms is attributable to glycosylation. Immunohistochemistry revealed SR-BI to be concentrated in the cytoplasm of follicular granulosa cells, although found mostly at the periphery of luteal cells. To examine receptor dynamics during gonadotropin-induced luteinization, pigs were treated with an ovulatory stimulus, and ovaries were collected at intervals to ovulation. SR-BI in granulosa cell cytoplasm increased through the periovulatory period, with migration to the cell periphery as the CL matured. In vitro culture of follicles with human chorionic gonadotropin induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. We conclude that luteinization causes upregulation of SR-BI expression, its posttranslational maturation by glycosylation, and insertion into luteal cell membranes. Expression of the LDL receptor is extinguished during luteinization, indicating dynamic regulation of cholesterol importation to maintain elevated steroid output by the CL.

ovary; gene expression; ovulation; granulosa cell differentiation

THE FORMATION OF THE corpus luteum (CL) after rupture and collapse of the ovulating follicle requires widespread cell and tissue remodeling, driven by extensive changes in gene expression (17). A significant aspect of luteal remodeling is the shift from estrogen as the principal steroid product to progesterone, a change accompanied by a large-scale increase in total steroid synthesis. At the peak of their output during the late follicular phase, estrogens are synthesized only in small amounts (µg/day), whereas the human CL secretes as much as 40 mg/day of progesterone during the luteal phase (13). To achieve this amplification in steroidogenesis, a major increase in the amount of the steroid parent molecule, cholesterol, is required. The quantitatively most important supply of cholesterol for luteal steroid synthesis arrives by importation from extracellular sources, in the form of high- and low-density lipoprotein (HDL and LDL)-borne cholesterol (3, 20).

Importation of lipoprotein cholesterol from LDL and HDL is mediated by specific membrane receptors. The endocytic LDL receptor system and attendant cholesterol importation have been abundantly characterized in a number of tissues, including the ovary (3, 20). In recent years, it has been shown that HDL importation depends on a cell surface glycoprotein known as scavenger receptor BI (SR-BI), a glycoprotein composed of two transmembrane and two cytoplasmic domains and a large N-glycosylated extracellular loop (24). The SR-BI gene codes for a protein with a predicted mass of 57 kDa, and immunoblot and immunoprecipitation experiments have revealed an apparent mass of 82 kDa (1), suggesting extensive glycosylation. SR-BI is inserted in the plasma membrane as a functional dimer where it serves as high-capacity bulk cholesterol delivery system (23).

Tissue profiles have demonstrated the highest expression of SR-BI in the ovary and adrenal of the rat (10), and in situ hybridization have revealed that expression is restricted to the theca compartment of developing follicles (24). In rat (11) and bovine (2) ovaries, transcript abundance appears to increase as follicular development ensues. In the rat ovary, SR-BI expression is upregulated by folliculogenetic stimuli, in particular, the combination of luteinizing hormone (LH) and insulin, which induces theca cell steroid synthesis (12). Antibody neutralization of SR-BI in rat theca cell cultures reduces gonadotropin-stimulated steroidogenesis by 90% (28), attesting to the dominance of SR-BI in cholesterol supply in this follicular compartment. In the rat, SR-BI expression becomes established with the remodeling of the ovary during luteinization, as first shown in vitro by Azhar et al. (4) and evidenced by a large-scale increase in expression of the SR-BI message in the developing CL (11). These changes are evident at 12 h after the ovulatory stimulus, around the time of ovulation. In vitro evidence supports a role for gonadotropin regulation of SR-BI, as treatment of rat luteal cells with the LH analog human chorionic gonadotropin (hCG) provokes both the expression and dimerization of the mature (82 kDa) form of SR-BI (23). Because little is known about kinetics of the changes in gene expression that accompanies the formation of the CL, we undertook exploration of the mechanisms of importation of cholesterol during the luteinization process by examining expression of the LDL receptor and SR-BI in the porcine ovary and by means of experimental paradigms to invoke luteinization in vivo and in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, tissues, and treatments. All animal experiments were approved by the University of Montreal Animal Care Committee and performed according to the Canadian Council of Animal Care regulations. To determine the temporal expression and cellular localization of SR-BI protein throughout the ovulatory process, 15 gilts were treated with 500 IU equine chorionic gonadotropin followed by 100 IU hCG (Intervet Canada Whitby) to induce ovulation. Ovaries were recovered at 0, 24, 30, and 38 h after hCG administration. In other trials, pig ovaries were collected at the slaughterhouse. Follicular fluid rich in granulosa cells was aspirated from small (<3 mm diameter), medium (4–7 mm), and large (>8 mm) follicles and washed three times before RNA and protein analyses were conducted. CLs were classified according to their developmental morphology (19) as postovulatory (CL-I), developing (CL-II), midluteal (CL-III), and regressing (CL-IV). To investigate SR-BI protein expression in situ, whole follicles between 3.5 and 5 mm in diameter were dissected from ovaries acquired at the slaughterhouse and cultured in Opti-MEM (Invitrogen, Carlsbad, CA) containing 1% insulin and 10% FBS (Invitrogen). At 12 h after initiation of incubation, hCG (100 or 500 IU) was added to the culture medium, and follicle cultures were terminated after a further 12 or 30 h of incubation. For follicle culture, granulosa cells were aspirated from three independent follicles and rinsed with culture medium to eliminate follicular fluid. We employed a further paradigm of in vitro luteinization of granulosa cells as previously described (22). Briefly, granulosa cells from of 3- to 5-mm-diameter follicles were aspirated from slaughterhouse ovaries and seeded at a rate of 7.5 million cells/ml of Opti-MEM containing 1% insulin and 10% FBS. Cells were treated with medium alone or medium plus 5 µg/ml actinomycin D (Sigma). After 24 h, medium was changed, and fresh medium or medium + 100 ng/ml LH (National Institutes of Health, Department of Agriculture) was added. Cultures were terminated 24 or 48 h later. In a second trial, freshly isolated granulosa cells were incubated in medium containing 10% FBS or 10% fetal bovine lipoprotein-depleted serum (FBLDS; GIBCO BRL) for 48 h. In both the whole follicle and isolated granulosa cell culture trials, results were based on three independent experiments from ovaries acquired on different days.

Pig SR-BI cloning, sequencing, and RT-PCR. Total RNA extracted from a midcycle porcine CL, quantified by spectrophotometry at 260 nm, and a 1-µg sample were used for RT with the Omniscript RT kit (Qiagen, Mississauga, ON) according to the manufacturer's instructions. SR-BI primers were designed based on a homologous sequence in GenBank (accession no. AF467889) as follows: TGA CTC CCG AAT CCT CTC TG (forward) and CTG CTC CTT GCT CTG GGCT (reverse). The consequent 550-bp PCR product was resolved on 1% agarose gel, excised, and purified with a gel extraction kit (Qiagen). The cDNA was ligated into a pGEM-T Easy Vector System I (Promega, Nepean, ON) according to the manufacturer's instructions and transformed into competent Escherichia coli strain XL-1 blue. Plasmids were isolated with a QIAprep Spin Miniprep kit (Qiagen) and sequenced (Applied Biosystems, Foster City, CA). Three independent samples were sequenced to verify authenticity. Cell pellet samples and ~5 mm3 of tissue from each CL were homogenized in buffer containing guanidine isothiocyanate (4.23 M) and 0.12 M beta-mercaptoethanol (both from Sigma, St. Louis, MO). RNA was purified with an RNeasy Protect mini kit (Qiagen) as recommended by the manufacturer. Pig-specific primers (forward: GTC CAT GCC ATC ACT GCC ACT TG; reverse: CCT GCT TCA CCA CCT TCT TG) for GAPDH were used as control. A preliminary investigation indicated that 27 PCR cycles in a final volume of 25 µl, using Taq DNA polymerase (Amersham Biosciences, Baie d'Urfe, QC) with a 58°C annealing temperature, addressed the linear portion of the amplification curve for both SR-BI and GAPDH. Semi-quantitative PCR amplification for SR-BI and GAPDH in luteal tissues and granulosa cells was performed with tissues from three to five animals, and products were resolved on 1% agarose gel using 1% ethidium bromide. Optical densities were analyzed with the Alpha-imager densitometer (Alpha Innotech, San Leandro, CA).

Western blot analysis. One milligram of tissue as described above was homogenized with 300 µl of protein loading buffer containing beta-mercaptoethanol and maintained at –20°C until analysis. Protein concentrations were determined by the Bradford (Bio-Rad) assay, and an aliquot of 30 µg of protein was loaded per well. Blots were electrophoretically separated by one-dimensional 10% SDS-PAGE at 105 V. High molecular weight standards (Amersham Pharmacia, Piscataway, NJ) were used as reference. Resolved proteins were transferred to a 0.45-µm nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) and stored overnight at 4°C. Membranes were washed with distilled water and stained with Ponceau solution, washed again in 0.1% Tween in Tris-buffered saline (100 mM Tris + 0.9% sodium chloride from Sigma, pH 7.5), and blocked with 1% BSA in 0.1% Tween in Tris-buffered saline. A polyclonal SR-BI first antibody (Novus Biological, Littleton, CO) was added at 1:1,000 final concentration, followed by 2-h incubation, and a second antibody, anti-rabbit IgG, labeled with horseradish peroxidase (Amersham Biosciences UK, Buckinghamshire, UK) was applied at 1:10,000 over 30 min. We further examined SR-BII, a splice variant of SR-BI, with the use of a polyclonal antibody (Novus Biological) at 1:1,000. The LDL receptor was evaluated by means of a monoclonal antibody (Abcam, Cambridge, MA) at 1:1,000 concentration incubated for 2 h, and a second antibody, rabbit anti-mouse IgG conjugated to horseradish peroxidase (EMD Biosciences, San Diego, CA), was used at 1:10,000 over 30 min. As a control, {alpha}-tubulin was probed with a monoclonal antibody developed by the Developmental Hybridoma Bank (Iowa City, IA) at 1:1,500 concentration under conditions of incubation for 2 h; the same second antibody and incubation time used for LDL receptors were applied. All incubations were carried out at room temperature. Membranes were rinsed, and abundance of all proteins was detected with chemiluminescent substrate (Pierce Biotechnology, Rockford, IL); blots were exposed to Kodak (Rochester, NY) photographic films. The optical density of the protein band was quantified by scanning with an Alpha-image densitometer.

Immunofluorescence. Ovaries, follicles, and CL were fixed in Zamboni solution, embedded in paraffin, and sectioned at 6 µm. Tissue sections were rehydrated and, for antigen recovery, boiled in 10 mM sodium citrate (pH 6.0) for 10 min. Slides were then rinsed once in cold (4°C) PBS (5 min), blocked for 20 min with 1% BSA, and incubated over 2 h at room temperature using the SR-BI polyclonal antibody at 1:50. Tissues were rinsed twice in PBS and incubated for 30 min with a second antibody labeled with a cyanine dye (Cy3: Biocan Scientific, Mississauga, ON). Nuclei were stained with 1:1,500 4'-diamidino -2-phenyindole dilactate (Sigma). All procedures were performed at room temperature. Slides were mounted in Permafluor (Thermo Shando, Pittsburgh, PA).

Deglycosylation of SR-BI. To establish the basis for SR-BI forms of variant molecular weight, CL-III tissues were subjected to in vitro deglycosylation using endoglycosidase H (Endo H) and N-glycosidase F (PNGase; both from New England Biolabs, Ipswich, MA) (0.8–1 mg) and lysed in 300 µl of protein loading buffer plus beta-mercaptoethanol (14.3 µl/ml); 50-mg aliquots of total protein (evaluated by Bradford) were denatured at 100°C over 5 min. Then, 1/10 volume of G5 buffer (0.5 M sodium citrate, pH 5.5 at 25°C) and 5 units of Endo H were added, and the mixture was incubated overnight at 37°C. For PNGase digestion, 50 mg of total protein were treated with 5 U of the glycosidase in beta-mercaptoethanol and >G7 buffer followed by overnight incubation at 37°C. The undigested sample (control) was incubated without enzyme. Samples were subjected to Western blot analysis in one-dimensional 8% SDS-PAGE at 105 V. High molecular standard weight protein markers (Amersham) were used as reference. Samples were evaluated in triplicate.

Statistical analyses. SR-BI mRNA optical density was standardized by comparison to GAPDH. For SR-BI protein expression, optical density data were transformed using square root to obtained normality of distribution; transformed data were analyzed by split-plot ANOVA. SR-BI protein deglycosylation was tested with {chi}2 procedures. LDL receptor and SR-BI protein optical density data were transformed by square root and analyzed with a 2 x 2 factorial design ANOVA. The level of significance for all analyses was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of SR-BI in granulosa cells from ovarian follicles and CL. RT-PCR analysis revealed that SR-BI mRNA was present in granulosa cells aspirated from all classes of ovarian follicles collected and did not differ in abundance with the stage of follicle development (Fig. 1). Luteal transcript abundance was consistently 1.5- to 2.0-fold greater and did not differ throughout the luteal phase of the estrous cycle (Fig. 1). SR-BI protein quantification of immunoblots indicated that granulosa cells of all follicles express both the 57- and 82-kDa forms of SR-BI (Fig. 2). The abundance of the lower molecular mass form differed from barely detectable in small follicles to more pronounced in CL. In contrast, the 82-kDa form, also barely detectable in follicles, is robustly expressed in CL. The relative abundance was consistent with the pattern of mRNA expression in that it was more strongly expressed in CL relative to follicles (Fig. 2). The total immunoreactive protein increased six- to sevenfold with luteinization, whereas the proportion of the 57-kDa form declined from an average of 10% in follicles to 5% in CL.


Figure 1
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  		Fig. 1. Follicles and corpora lutea (CL) express scavenger receptor-BI (SR-BI) transcripts. A: representative RT-PCR analysis revealing the occurrence and relative abundance of SR-BI transcripts in small follicles (SF; <3 mm diameter), medium follicles (MF; 4–7 mm), large follicles (LF; >8 mm), and CL across the progression of the luteal cycle (CL-I to CL-IV). GAPDH was used as a control. B: means ± SD of SR-BI transcript abundance relative to GAPDH for 5 follicles and 5 CL at each stage of development. Means bearing different superscripts are significantly different (P < 0.05).

 

Figure 2
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  		Fig. 2. Developmental sequence of SR-BI protein abundance in ovarian tissues. A: representative immunoblot demonstrating the 2 species of protein (82 and 57 kDa) detected in porcine ovarian tissues. Tubulin served as loading and constitutive control. B, top: means ± SD of the relative abundance of the 82-kDa version SR-BI in a series of 5 follicles and 5 CL at each stage of development. B, bottom: means ± SD relative abundance of the 57-kDa form of SR-BI relative to tubulin. Means with different superscripts are significantly different (P < 0.05).

 
Subsequent experiments were undertaken to determine whether the difference between the 82- and 57-kDa forms was related to the carbohydrate moiety of the glycoprotein. Figure 3 depicts the effects of digestion of protein extracts of midcycle CL (CL-III) with Endo H, a glycosidase that cleaves mannose and some hybrid oligosaccharides from N-linked glycoproteins, and with PNGase, an amidase that cleaves N-linked carbohydrates between the innermost GlcNAc and asparagine residues (14). Endo H treatment reduced the 82-kDa form substantially, to a consequent 68-kDa form, with no apparent effects on the 57-kDa version, suggesting partial deglycosylation (Fig. 3). In samples subjected to PNGase digestion, the 82-kDa form was entirely eliminated, and only the 57-kDa form persisted (Fig. 2). Together, these digestions indicate that the difference between the two forms of SR-BI in CL (Fig. 2A) is based on glycosylation of the 82-kDa species.


Figure 3
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  		Fig. 3. Effects of enzymatic deglycosylation on SR-BI from CL as revealed by immunoblot. An aliquot of 50 µg of luteal protein extracts was digested overnight at 37°C with endoglycosidase H (Endo H) or N-glycosidase F (PNGase). Top: representative immunoblots demonstrating the relative abundance of the products of digestion. Tubulin was used as a control for loading. Bottom: percentages of the total sample found in each molecular mass category between control and digested protein samples for each of the enzyme treatments (representative of 3 replications of the trial).

 
Changes in cholesterol importation receptor abundance during the periovulatory and luteinization periods. Immunolocalization was employed to further characterize the differences in cholesterol transport mechanisms between follicular and luteal cells. As depicted in Fig. 4, the SR-BI signal is present throughout the cytoplasm in theca and granulosa cells but is restricted to the periphery of luteal cells. Based on this observation, we examined the changes of SR-BI abundance and localization during the ovulatory process in the pig (Fig. 5). In the largest follicles of untreated pigs, SR-BI distribution conformed to the observations in Fig. 4, with cytoplasmic expression in the theca and granulosa cells, the latter much reduced compared with the former. The first 24 h after the ovulatory stimulus were characterized by a modest increase in cytoplasmic SR-BI expression in granulosa cells that became more intense as ovulation approached (30 and 38 h, Fig. 5A). Early CL (CL-I, 24–48 h after ovulation) displayed both cytoplasmic and membrane localization of SR-BI; however, in later stages, the signal was most commonly found on the periphery of the cells (Fig. 5B).


Figure 4
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  		Fig. 4. Immunocytochemistry demonstrating the divergent cellular localizations of SR-BI in cells from compartments of the porcine ovary. Photomicrographs depict representative sections of 3- to 5-mm follicles showing the theca and granulosa component, along with a representative section from a midcycle CL. Indirect localization was used, with cyanine dye (Cy3) as the fluorescent signal for SR-BI and 4'-diamidino-2-phenyindole dilactate (DAPI) as a nuclear marker.

 

Figure 5
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  		Fig. 5. Changes in cellular localization of SR-BI during the periovulatory and luteal phases of the estrous cycle in the pig. A: representative sections demonstrating SR-BI expression and localization in follicles at 0, 24, 30, and 38 h after human chorionic gonadotropin (hCG) administration. As in Fig. 4, the Cy3 marker indicates SR-BI localization and DAPI the nuclear signal. B: changes in the SR-BI signal across the course of luteal development and during luteal regression in representative CL, classified according to morphological characteristics. T, theca cells; G, granulosa cells; BM, approximate localization of the basement membrane separating the theca and granulosa compartments.

 
There are multiple mechanisms of cholesterol supply to steroidogenic tissues. Perhaps the second most significant in the ovary is LDL receptor-mediated importation (15). It was therefore of interest to establish whether concurrence, complementarity, or some other mechanism characterized the cholesterol import receptors during transformation of the porcine follicle into the CL. Immunoblotting revealed that the LDL receptor was highly expressed in granulosa cells of small, medium, and large follicles (Fig. 6, A and B), but the signal became undetectable with luteinization. Concurrent analysis of SR-BI demonstrated a pattern of expression identical to that displayed in Fig. 2 (Fig. 6, A and B), such that the 82-kDa form was virtually absent from follicles and was acquired by the CL after ovulation. Together, these results indicate a temporal sequence in expression of the receptors and complementarity between the two extracellular cholesterol supply processes during the process of CL formation.


Figure 6
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  		Fig. 6. Comparison of the abundance of the low-density lipoprotein receptor (LDL-R) and SR-BI in follicles and CL by immunoblot. Pig follicles and CL were classified as in Fig. 1. A: representative Western blot demonstrating the occurrence of LDLR in the follicular granulosa cells but not in CL; SR-BI was found, as in previous figures, primarily in CL. Tubulin expression was used as loading control. B: mean density of protein bands showing reciprocal expression of the LDLR (high in follicles, low in CL; P < 0.01) and SR-BI (low in follicles, high in CL; P < 0.01) in tissues representing the events of folliculogenesis and the luteal phase in the pig. C: immunoblots representative of analysis of porcine CL across the luteal phase demonstrating the relative abundance of SR-BII, a splice variant of SR-BI.

 
Alternative splicing of the SR-BI transcript results in a second form of the protein with a different COOH-terminal cytoplasmic tail, apparently conferring predominant intracellular expression (5). Because this splice variant represents some 10–15% of SR-BI in the liver (27), we examined its abundance through the luteal phase by immunoblotting. The results (Fig. 6C) demonstrate that this isoform is present in the CL but that its relative abundance does not vary across the luteal phase of the estrous cycle.

Gonadotropin induction of the pattern of cholesterol importation in vitro. We then addressed the mechanism by which these modifications in the cholesterol acquisition system might be controlled. We first employed an in vitro model of follicular function in which whole follicles were placed in organ culture for periods up to 30 h. Granulosa cells harvested from follicle culture under conditions expected to induce luteinization, i.e., the presence of 10% serum, displayed the expected low level of SR-BI and elevated LDL receptor protein abundance (Fig. 7). In granulosa cells aspirated from follicles incubated with serum, but not treated with gonadotropin, there was an increase in SR-BI expression over 12 h relative to freshly isolated granulosa cells. Treatment with the ovulatory stimulus, in the form of 100 and 500 IU hCG, provoked significant increases in the expression of this receptor in the granulosa cell compartment (Fig. 7). These changes persisted through 30 h of treatment. In contrast, and as expected, LDL receptor abundance was greatest in granulosa cells aspirated from newly collected follicles (Fig. 7). Significant declines were induced by both doses of hCG at 12 h, and these persisted through 30 h, recapitulating the observations of luteinization in vivo.


Figure 7
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  		Fig. 7. Induction of the reciprocal expression of the LDLR and SR-BI in granulosa cells from porcine follicles in culture. Whole follicles between 3.5 and 5 mm in diameter were dissected from ovaries, incubated for 12 h, followed by addition of hCG (100 or 500 IU), and incubated until termination of the cultures at 12 or 30 h after initiation of hCG treatment. The granulosa cells were aspirated and subjected to Western analysis. A: representative immunoblot. B and C: means ± SD of abundance of each receptor relative to tubulin in triplicate incubations. Means bearing different superscripts are significantly different (P < 0.05).

 
To complement whole follicle studies and to determine to what extent the increases in SR-BI expression were related to posttranscriptional changes, the model of transcriptional inhibition with actinomycin D during in vitro luteinization of isolated granulosa cells (18) was employed. In cells incubated with LH, there was an approximate doubling in SR-BI transcript abundance at 24 and 48 h relative to untreated control (Fig. 8A). A similar increase in protein was observed at 24 h, although there was an approximately fivefold augmentation at 48 h (Fig. 8B). Actinomycin D treatment (Fig. 8) obliterated both the mRNA and protein signals, indicating that increases in abundance of the message and protein were due to new transcription, rather than to posttranscriptional regulation. Progesterone analysis (Fig. 8C) demonstrated that LH induced increases in the accumulation of this steroid at 24 h relative to control, an effect that was abrogated by actinomycin D treatment. At 48 h, progesterone accumulation was increased in both control and LH-treated cultures. There was substantial accumulation in the actinomycin D-treated cells, indicating that the cells remained alive and steroidogenic, despite absence of significant amounts of SR-BI.


Figure 8
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  		Fig. 8. Induction of SR-BI expression in isolated luteinized porcine granulosa cells with 100 ng/ml luteinizing hormone (LH) and the effects of transcriptional blockade with actinomycin D (Act-D). Granulosa cells isolated from 3- to 5-mm porcine follicles were treated with culture medium alone or medium plus 5 µg/ml Act-D. After 24 h, medium was changed and fresh medium or medium + 100 ng/ml LH (NIH USDA) was added. Cultures were terminated 24 or 48 h later, and SR-BI transcript (A) and protein (B) abundances were determined. Progesterone accumulation in culture medium was determined by radioimmunoassay (C). Bars represent means ± SD from 3 replicate experiments. Means bearing different superscripts are significantly different (P < 0.05).

 
We also examined whether lipoprotein content of the medium influenced the expression of SR-BI by incubation with either FBS or FBLDS. The results (Fig. 9) indicate that, although there was an overall accumulation of progesterone when FBLDS was present, relative to FBS, there was no evidence that lipid-depleted serum affected SR-BI expression, in either transcript or protein abundance.


Figure 9
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  		Fig. 9. Effects of lipoproteins in culture medium on expression of SR-BI by luteinized porcine granulosa cells. Cells, isolated from 3- to 5-mm porcine follicles, were cultured for 48 h in medium containing 10% FBS or 10% fetal bovine lipoprotein-depleted serum (FBLDS). SR-BI expression was determined by PCR amplification for transcript abundance (A) and immunoblot analysis for protein expression (B). Progesterone accumulation in medium was determined by radioimmunoassay (C). Data are means ± SD from 3 replicate experiments. Means with different superscripts are significantly different (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The mammalian CL is the product of extensive tissue remodeling of the ovarian follicle that begins with ovulatory stimulus. Its presumed evolutionary advantage, and thus its function, is the large-scale secretion of the progestins required for viviparity. The principal source of cholesterol substrate for this steroid output is importation from extracellular sources (15, 20). The goal of this investigation was to examine the process of luteinization, in terms of the changes in cholesterol importation mechanisms.

Previous studies in the rat have suggested that, although SR-BI is present in the theca compartment, it is absent from the granulosa cells of ovarian follicles (12). In contrast, we found transcripts to be present and low levels of protein detectable in granulosa cells of all classes of follicles from the porcine ovary. Immunocytochemical observations in the present study indicated that the localization of SR-BI expression in granulosa cells is, at least in part, cytoplasmic, even up to the period of periovulatory differentiation. This distribution is surprising, and perhaps counterintuitive, given the high concentrations of HDL in porcine follicular fluid (7) and the indication that the function of SR-BI as the HDL receptor requires its insertion in the plasma membrane (24). It is known that porcine granulosa cells do not synthesize steroids from cholesterol, due to absence of steroidogenic acute regulatory protein before the onset of luteinization (8, 22) and low-level expression of cytochrome 450 side-chain cleavage (6). Together, these observations suggest that HDL uptake is not an essential element of follicular granulosa cell function.

Our findings demonstrate that, in the pig ovary, there are substantial increases in abundance of both SR-BI message and protein associated with luteinization. Qualitative immunohistochemical observations suggest that this is a gradual change, beginning during the first 24 h after the ovulatory stimulus and continuing through luteinization. The molecular and microscopic data in the porcine model concur with observations of in situ hybridization findings in the rat ovary, where there is a progressive increase in signal abundance and intensity as the luteinization process ensues (11), and with studies of rat granulosa cells in vitro where SR-BI transcript and protein both gradually increase during luteinization (4). The latter authors demonstrated that SR-BI expression was essential for the uptake of esterified cholesterol by luteinized granulosa cells (4).

Functional luteal regression, i.e., the reduction in steroidogenic capacity, precedes structural regression in most species (25). The continued expression of SR-BI in regressed CL observed in the present investigation suggests uncoupling of the processes of luteal steroidogenesis and the cholesterol intake at the end of the luteal phase. This is in contrast to intracellular steroid transport mechanisms, including the steroidogenic acute regulatory (21) and the Niemann-Pick C-1 proteins (9), which decline rapidly after treatment with a luteolytic stimulus.

From these findings, we infer that, during the process of granulosa cell remodeling into large cells of the CL (19), the SR-BI signal is translocated to the cell periphery and presumably to the cell surface at a site in which it can function in cholesterol exchange. There is a large-scale increase in the quantity of the 82-kDa form of the protein accompanying luteinization, including a doubling in its abundance during the progression from the postovulatory (CL-I) to the midcycle (CL-II and III) CL. Follicle and isolated cell culture experiments indicated that the expression of SR-BI is induced by gonadotropin treatment, arguing that this event is specific to the luteinization process. The proportion of total immunoreactivity in the larger molecular mass form was double in luteal cells compared with granulosa cells, indicating that differentiation involves an alteration in or upregulation of the glycosylation process. This is at odds with the observation that N-glycosylated residues are cotranslationally incorporated into the protein during its synthesis (24) but may be a further reflection that the granulosa cell version of the receptor is nonfunctional before the ovulatory stimulus. Confirmation requires further investigation.

A further interesting finding is that HDL and LDL receptors display a complementary pattern of expression during ovarian follicle remodeling. The granulosa cell expression of the LDL receptor is marked and invariable in the developing follicle despite the apparent absence of LDL in porcine follicular fluid (7). The present investigation indicates that LDL receptor is lost during luteinization and that the signal is virtually undetectable in the CL. The pattern of SR-BI expression is reciprocally related to that of the LDL receptor, at lowest levels in follicle granulosa cells and highest in the CL, despite elevated concentrations of HDL in follicular fluid (7).

The question that arises is whether the dramatic switch in cholesterol uptake from LDL receptor to SR-BI is essential or whether the two importation strategies are redundant in CL function. Mice bearing an inactivating mutation of the SR-BI gene, although infertile, are capable of forming morphologically functional CLs that produce peripheral progesterone at concentrations that do not differ from their wild-type counterparts (26). Ovaries from SR-BI-null mice transplanted to ovariectomized, immunocompromised mice supported ovulation and gestation (16), further indicating normal luteal function. In fact, sterility in these animals has been recently attributed to dyslipedemia, resulting in the absence of normal bidirectional flux of HDL in the liver (30), rather than to luteal insufficiency. Nonetheless, there are noticeable differences between the SR-BI knockout and wild-type mice in the Oil-Red-O staining of the CL, indicating reduction in intracellular cholesterol stores in the former (26). Recent studies have suggested that HDL-borne cholesterol and cholesterol derived from de novo synthesis contribute approximately equally to the steroid substrate pool in the mouse ovary, whereas LDL cholesterol has only a minor role in ovarian steroidogenesis (29). It is not clear whether de novo cholesterol synthesis or LDL receptor-mediated importation compensates for the absence of the HDL cholesterol delivered via SR-BI in the mouse.

Less is known about the relative contribution of cholesterol sources in other species. Herein, we presented evidence to demonstrate that SR-BI, the HDL receptor, is strongly expressed during luteinization and that this expression is provoked by ovulatory stimuli. Furthermore, in vitro studies showed that SR-BI is induced by gonadotropins through new gene transcription, and it appears that this process is not influenced by lipoprotein availability. This implicates HDL cholesterol as the major source of substrate for luteal steroidogenesis in the pig.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The work was supported by Operating Grant MOP-11018 to B. D. Murphy from the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We thank Mira Dobias-Goff for valuable technical assistance, Vickie Roussel and Catherine Dolbec for aid with the figures, and Adrian Quero for input into experimental procedures and analysis of data.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. D. Murphy, Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, St-Hyacinthe, Québec, Canada J2S7C6 (e-mail: bruce.d.murphy{at}umontreal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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