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research-article |
Centre de Recherche en Reproduction Animale,3 Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
Max F. Perutz Laboratories,4 Department of Medical Biochemistry, Medical University of Vienna, A-1030 Vienna, Austria
ABSTRACT
The low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) is a member of the LDL receptor family that participates in endocytosis and signal transduction. We cloned the full-length bovine LRP8 cDNA in granulosa cells (GC) of the dominant follicle (DF) as well as several LRP8 mRNA splicing variants, including a variant that contains a proline-rich cytoplasmic insert (A759-K817) that is involved in intracellular signaling. Expression of the A759-K817 variant was analyzed in the GC of follicles at different developmental stages: the small follicle (SF; 2–4 mm), the DF at Day 5 (D5) of the estrus cycle, ovulatory follicles (OF) 24 h after hCG injection, and corpora lutea (CL) at D5. RT-PCR analysis showed that expression was predominant in the GC of DF compared to other follicles and CL (P < 0.0001), whereas the expression of other related receptors, such as LDLR and VLDLR, did not show differences. Temporal analyses of follicular walls from the OF following hCG treatment revealed a decrease in LRP8 mRNA expression starting 12 h post-hCG treatment (P < 0.0001). LRP8 protein was exclusively localized to the GC, with higher levels in the DF than in the SF (P < 0.05). RELN mRNA, which encodes an LRP8 ligand, was highly expressed in the theca of the DF as compared to the OF (P < 0.004), whereas MAPK8IP1 mRNA, which encodes an LRP8 intracellular interacting partner, is expressed in the GC of the DF. These results demonstrate the differential expression patterns of LRP8, RELN, and MAPK8IP1 mRNAs during final follicular growth and ovulation, and suggest that a RELN/LRP8/MAPK8IP1 paracrine interaction regulates follicular growth.
follicle, follicular development, granulosa cells, ovulation
During the bovine estrus cycle, two or three sequential waves of follicular development are observed, each producing a dominant follicle that is capable of ovulating if luteal regression occurs. Each follicular wave is preceded by an increase in the circulating FSH concentration, which initiates the recruitment of 7–11 antral follicles to grow larger than 3–4 mm in diameter [1, 2]. With declining FSH concentrations, the number of growing follicles is reduced until only one follicle is selected to continue its development to becoming the dominant follicle (DF); meanwhile, subordinate follicles degenerate by apoptosis. The DF is responsible for the maintenance of FSH concentrations below the threshold requirements of smaller follicles through increased secretion of estradiol and inhibin, which negatively regulate FSH secretion [2, 3]. At the basal circulating FSH concentration, the DF ensures its growth by the expression of molecular determinants, such as the LH/choriogonadotropin receptor (LHCGR), cytochrome P450, members of family 19 subfamily A, polypeptide 1 (CYP19A1), growth factors that belong to the inhibin family, and members of the insulin-like growth factor system that synergize with the actions of gonadotropins. These factors have been shown to be differentially expressed in granulosa cells (GC) at the mRNA and protein levels in relation to the development of the DF [2, 4–7]. Despite the identification of many important factors, the mechanisms that allow a DF to be selected, to pursue its growth in the presence of a low circulating FSH concentration, and potentially to ovulate, are still not fully understood.
The identification of genes that are upregulated in the GC of growing DF compared to their expression in nonselected small follicles (2–4 mm) has been accomplished by suppression subtractive hybridization (SSH) [6], and it has allowed the isolation of a differentially expressed cDNA fragment that corresponds to bovine low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2). A similar study identified LRP8 as being preferentially expressed in bovine DFs rather than in subordinate follicles [8]. LRP8 is a member of the low-density lipoprotein (LDL) receptor protein family, which includes the LDL receptor (LDLR) and the very-low-density lipoprotein receptor (VLDLR). These cell surface receptors are known to mediate the uptake of lipid-rich extracellular lipoproteins into the cell, and they have also been shown to transduce extracellular signals and activate intracellular tyrosine kinase signaling [9, 10]. Several splicing variants of LRP8 mRNA have been detected and are predicted to encode proteins that lack regions of the extracellular ligand-binding domain, the threonine- and serine-rich domain, and the 59-amino acid, proline-rich, cytoplasmic insert that interacts with the mitogen-activated protein kinase 8-interacting protein 1 (MAPK8IP1; also known as the c-Jun N-terminal kinase-interacting protein 1, JIP-1) [11, 12]. At present, the functional significance of these splicing variants is not fully understood [13–16]. Extracellular binding of the natural ligand of LRP8, reelin (RELN), results in the phosphorylation of an intracellular protein, disabled-1 (DAB1), and the activation of downstream signaling pathways. Defects in these signaling cascades, as in lrp8-deficient mice, cause developmental defects, such as abnormal layering of neurons in the cortex, hippocampus, and cerebellum [17, 18], and learning deficiency in adults [16]. In humans, the expression of LRP8 mRNA has been detected in the brain and placenta, whereas in rabbits, expression has been found in the brain and testis, and to a much lesser extent, in the ovary [13]. Male lrp8-deficient mice exhibit infertility and lrp8 is required for sperm maturation [18, 19]. Since an mRNA expression profiling study [6] has identified a cDNA fragment that corresponds to LRP8 in the GC of bovine DFs, we hypothesized that the expression of LRP8 is associated with follicular dominance. The objectives of this study were to clone the corresponding full-length bovine LRP8 cDNA in GC, to analyze the expression of potential LRP8 mRNA isoforms, to study the spatio-temporal expression profiles of the LRP8 mRNA and protein using an in vivo model of bovine follicles collected at different developmental stages, and to explore the expression of LRP8-interacting proteins, such as RELN, MAPK8IP1, MAPK8IP2, and DAB1, in dominant follicles.
Animal Model, Tissue Collection and RNA Isolation
Bovine ovarian follicles and corpora lutea (CL) were isolated at specific stages of the estrus cycle from normal cyclic crossbred heifers that were held at the large animal complex of the Faculty of Veterinary Medicine, as previously described [6, 20]. Briefly, estrus cycles were synchronized with PGF2
(25 mg Lutalyse i.m.; Upjohn, Kalamazoo, MI) and ovarian follicular development was monitored by daily transrectal ultrasonography until ovariectomy. Based on ultrasonographic measurements, DFs (n = 4 cows) were defined as being larger than 8 mm and growing, while subordinate follicles were either static or regressing. DFs were obtained by ovariectomy (via colpotomy) on the morning of Day 5 (D5) of the estrus cycle (D0 = day of estrus). Ovulatory follicles (OFs) were obtained following injection of 25 mg PGF2 on D7 of the synchronized estrus cycle to induce luteolysis, thereby maintaining the development of the DF of the first follicular wave and transforming it into a preovulatory follicle. An ovulatory dose of hCG (3000 IU APL, i.v.; Ayerst Lab, Montréal, QC) was injected 36 h after the induction of luteolysis, and the ovaries that carried hCG-induced OFs were collected by ovariectomy at 0, 6, 12, 18, and 24 h after hCG injection (n = 2–4 cows/time-point). Follicles were dissected into preparations of follicular wall (theca interna with attached GC) [20] or further dissected into separate isolates of GC and theca cells (TC) [6]. Ovariectomies were also performed on D5 of the synchronized estrus cycle to obtain CL (n = 3 cows). In addition, GC were collected from 2–4 mm follicles obtained from slaughterhouse ovaries, representing a total of three pools of 20 small follicles (SFs). These experiments were approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine of the Université de Montréal. Total RNA was isolated from tissues as previously described [21]. The concentration of total RNA was quantified by measuring the optical density at 260 nm, and quality was evaluated by visualizing the 28S and 18S ribosomal bands following electrophoretic separation on a formaldehyde-containing denaturing agarose gel.
Cloning of the Bovine LRP8 cDNA
Three strategies were used to clone the bovine LRP8 cDNA. First, the size of the full-length bovine LRP8 cDNA was estimated at 4 kb from a previous virtual Northern blot analysis [6]. Thus, to construct the size-selected cDNA library, total RNA was first isolated from the GC of DFs obtained at D5 of the estrus cycle, and then transformed into cDNA by the SMART PCR cDNA synthesis method (BD Biosciences Clontech, Mississauga, ON) [6]. The cDNAs were size-fractionated by agarose gel electrophoresis, and cDNAs of between 3.5 kb and 4.5 kb were purified to construct a size-selected cDNA library based on the pDrive plasmid (Qiagen PCR cloning kit; Qiagen, Mississauga, ON), as described previously [22]. The cDNA library was then screened by radioactive hybridization [22] with a 500-bp bovine LRP8 probe, which was generated from a previous SSH screening experiment [6]. Positive LRP8-hybridizing bacterial colonies were grown, their plasmid content was isolated (QIA-prep; Qiagen), and the sizes of the cloned cDNAs were analyzed following EcoRI digestion and gel electrophoresis. The cDNAs were sequenced via the dideoxy sequencing method (Big Dye Terminator 3.0, ABI Prism; Applied Biosystems PE, Branchburg, NJ) and analyzed on an ABI Prism 310 sequencer (Applied Biosystems). Nucleic acid sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) and the GenBank database.
Since the bovine LRP8 cDNA obtained was incomplete at the 5'-end, the 5'-RACE system version 2.0 (Invitrogen, Burlington, ON) was used as directed by the manufacturer. Reverse transcription was performed using 2 µg of total RNA isolated from the GC of DFs, together with the bovine LRP8 antisense primer (5'-ATGGGCTTCTGCCAGCAACGG-3'). After terminal deoxynucleotidyl transferase tailing, the first PCR was performed with the sense abridged anchor primer (5'-RACE system; Invitrogen) and the bovine LRP8 antisense primer (5'-TAGCCAGGGTGGCACTCGC-3'). The second nested PCR reaction was performed with the sense universal amplification primer (5'-RACE system) and the bovine LRP8 antisense primer (5'-TCTGGGTCCTCGCACTCATCG-3'). PCRs were performed with Advantage 2 DNA polymerase (BD Biosciences Clontech) for 35 cycles (first reaction) or 30 cycles (second reaction) of 95°C for 1 min, 65°C for 30 sec, and 68°C for 1 min. The largest 5'-RACE product was isolated, subcloned into pDrive (Qiagen), and sequenced as described above. The sequence obtained contained only part of the missing 5'-end of the cDNA.
Finally, a bovine genomic library (BD Biosciences Clontech) was screened by a phage plaque hybridization procedure with a 1-kb LRP8 cDNA fragment that was labeled with [-32P]dCTP (NEN Life Sciences, Boston, MA) using the random priming method (T7 Quickprime; Amersham Biosciences, Pointe-Claire, QC). From the 6 x 105 phage plaques screened, one positive clone was purified through subsequent rounds of screenings. Recombinant DNA lambda phages were produced and purified [21], the DNA inserts were characterized by SacI restriction digestion, and the DNA fragments were subcloned into the pDrive vector. The nucleotide sequence of the clone was determined using specific oligonucleotide primers and shown to contain the complete 5'-end of the bovine LRP8 open reading frame (ORF) as well as the 5'-untranslated region (UTR).
Semiquantitative RT-PCR and Southern Blot Analyses
The expression patterns of specific genes were studied by semiquantitative RT-PCR in GC and TC preparations collected from follicles at different stages of development and from CL. Total RNA (1 µg) from GC, TC or CL was reverse-transcribed with an oligo(dT)30 primer and PowerScript (BD Biosciences Clontech), to generate the first-strand cDNA, using the SMART PCR cDNA synthesis kit [6]. Second-strand cDNAs were produced with the SMART II 5'-anchored oligonucleotide, and PCR-amplified for 18 cycles using Advantage 2 DNA polymerase. To perform semiquantitative RT-PCR, 1-µl aliquots of the SMART cDNA pools were used in a 25-µl PCR reaction with the Advantage 2 DNA polymerase kit. Gene-specific PCR primers were designed for the ORFs of the following bovine cDNA sequences: 1) the LRP8 mRNA variant that contains the proline-rich cytoplasmic insert A759-K817 (sense, 5'-CAGCTTTGATCACCCACTGTGGG-3' and antisense, 5'-GACTCAAGGACAGAAACCCTTCC-3'; AY364441); 2) LDLR (sense, 5'-TTGCCAGCCAAGCGGACACGG-3' and antisense, 5'-TTGCTGCCTTTGAGTGACTGGC-3'; XM_580492); VLDLR (sense, 5'-GTGACCACAGCAGTGTCGGAGG-3' and antisense, 5'-GCTGCTGGCTTGGTTACCATTCC-3'; NM_174489); RELN (sense, 5'-GTGGGCAGTTGACGACATCATC-3' and antisense, 5'-GATTGGCACACCCAATGTTAAGC-3'; AY568568); MAPK8IP1 (sense, 5'-TACGACTCGGTCAAGTATACGCTG-3' and antisense, 5'-AGCTCGAGCTCGTCTTCATGTCG-3'; XM_603140); MAPK8IP2 (sense, 5'-CTGCTCAGATGAGGACGACGAC-3' and antisense, 5'-TCCTCCTCAGCCTCCACTAGCAC-3'; XM_871210); DAB1 (sense, 5'-CTTCAGCATCATCATGCTGTTCACG-3' and antisense, 5'-GTAGAGAACCACACACGTCTGCAC-3'; BC067445); and the glyceraldehyde-3-phosphate dehydrogenase gene GAPD (sense, 5'-TGTTCCAGTATGATTCCACCCACG-3' and antisense, 5'-CTGTTGAAGTCGCAGGAGACAACC-3') [6]. For LRP8, LDLR, VLDLR, RELN, MAPK8IP1, MAPK8IP2, DAB1, and GAPD, the PCR conditions were as follows: an enzyme activation step at 95°C for 1 min, followed by a gene-specific optimized number of PCR cycles that corresponded to 95°C for 30 sec, 64°C for 45 sec, and 68°C for 90 sec. PCR was conducted for 23, 27, 27, 37, 38, 40, 40, and 18 cycles for LRP8, LDLR, VLDLR, RELN, MAPK8IP1, MAPK8IP2, DAB1, and GAPD, respectively. The PCR reactions were resolved in a 2% Tris-acetate-EDTA-agarose gel (40 mM Tris-acetate [pH 8], 1 mM EDTA) with ethidium bromide (0.5 µg/ml). The PCR products were visualized by UV and the images were digitized. The digitized signals for each gene obtained by semiquantitative RT-PCR were analyzed by densitometry using the ImageQuant software version 1.1 (Molecular Dynamics, Amersham Biosciences).
The OneStep RT-PCR system (Qiagen) was used for semiquantitative RT-PCR/Southern blotting [20] of LRP8 and GAPD (control gene) mRNA levels in bovine tissues. Total RNA samples were isolated from various tissues collected at a slaughterhouse, as described previously [21], and gonadotropin-stimulated GC were collected on D3 preceding PGF2 of a 4-day gonadotropin ovarian stimulation treatment [23]. Reactions were performed as directed by the manufacturer, using primers specific for bovine LRP8 (sense, 5'-TAAGCCTTGAAGATGATGGACTGC-3' and antisense, 5'-ATGCACACAGAGACCACATCATGG-3'; AY364441) and GAPD, as described above. Each reaction was performed using 100 ng of total RNA, and PCR was conducted for one cycle of 50°C for 30 min and 95°C for 15 min, followed by a variable number of PCR cycles of 95°C for 1 min, 64°C for 1 min, and 72°C for 1 min. The number of cycles used was optimized for each gene so as to fall within the linear range of PCR amplification, with 22 and 21 cycles for LRP8 and GAPD, respectively. Following PCR amplification, the samples were electrophoresed on 2% Tris-acetate-EDTA-agarose gels, transferred to nylon membranes, and hybridized with the corresponding radiolabeled LRP8 and GAPD cDNA fragments, as described previously [21]. The membranes were exposed to a phosphor screen, and signals were quantified on a Storm Imaging system using the ImageQuant software version 1.1 (Molecular Dynamics, Amersham Biosciences).
Alternative Splicing Analyses of Bovine LRP8 mRNA
The OneStep RT-PCR system was used to analyze the presence of alternative splicing variants of LRP8 in the GC of bovine DFs obtained at D5 of the estrus cycle. The reactions were performed as directed by the manufacturer. To amplify regions of interest, we used pairs of specific primers for the bovine LRP8 gene that corresponded to the extracellular segment of human LRP8 from exon 1 to exon 7 (exon 1: sense, 5'-GATCCACTGCACGGCGGCCAAG-3'; exon 2: sense, 5'-GATAACAGTGACGAGGACGACTGC-3'; exon 3: sense, 5'-ATGGCGAGGAGGACTGTCCAGAC-3'; exon 7: antisense, 5'-CTGAACGAGTGTCTGCACAACAATG-3'), the LRP8 O-linked sugar domain (sense, 5'-TGACATGAAGAGGTGCTACCG-3' and antisense, 5'-TGGGTAGACATGGCCAATCTGAGC-3'), and the LRP8 cytoplasmic segment (sense, 5'-ATGGGAATGAAGATGGAAAGATGGG-3' and antisense, 5'-GACTCAAGGACAGAAACCCTTCC-3'; AY364441). Each reaction was performed using 100 ng of total RNA from the GC of the DF, and PCR was conducted for one cycle of 48°C for 45 min and 94°C for 2 min, followed by a variable number of PCR cycles of 94°C for 30 sec, 64°C for 1 min, and 68°C for 2 min. The PCR products were size-resolved by electrophoresis on a 1.5% Tris-acetate-EDTA-agarose gel and visualized under UV light following ethidium bromide staining.
Cell Extracts and Immunoblot Analysis
Total protein extracts from bovine GC or CL were homogenized in M-PER buffer (Pierce, Rockford, IL) that was supplemented with a mix of protease inhibitors (Complete; Roche Applied Science, Laval, QC) as recommended by the manufacturer. Complete lysis of GC was achieved by multiple passages through a 25G needle attached to a 3-ml syringe. The CL were homogenized at 7000 rpm with a polytron PT1300D (Kinematica AG, Littau-Lucerne, Switzerland). The protein extracts were centrifuged at 16 000 x g for 15 min at 4°C, and the recovered supernatant (whole cell extract) was stored at –80 °C until electrophoretic analyses were performed. The protein concentration was determined by the Bradford method (Bio-Rad Protein assay; Bio-Rad Laboratories, Hercules, CA). Protein extracts (50 µg protein/sample) were heat-treated (5 min, 100°C), size-fractionated on a one-dimensional 5–20% gradient SDS-PAGE (Invitrogen), and electrophoretically transferred onto polyvinylidene difluoride membranes (PVDF, Hybond-P; Amersham Biosciences). Immunoblotting was performed as described previously [24]. The membranes were incubated with a rabbit polyclonal antibody raised against the complete intracellular domain of mouse LRP8 (1:5000) [25]. The expression of -glutathione S-transferase-1 (GSTA1) was used as a control, and the membranes were incubated with a rabbit polyclonal antibody against bovine GSTA1, as described previously [26]. Immunoreactive proteins were visualized by incubation with an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:15 000; Sigma-Aldrich, Oakville, ON) using NBT/BCIP as the alkaline phosphatase substrate (Roche Applied Science). The images were digitized using a ScanMaker 9800XL flatbed scanner (Microtek Laboratories, Redondo Beach, CA).
Immunohistochemical staining was performed on PBS-buffered formalin-fixed tissues, as described previously [24]. Paraffin-embedded tissues were cut to 3-µm thickness, mounted on SuperfrostPlus slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, and then rehydrated. Antigenicity lost during the fixation process was retrieved by heat treatment in a pressure cooker for 14 min, as described previously [24]. Non-specific binding sites were saturated by a 30-min incubation in the Tris-buffered saline blocking solution, i.e., TBS (100 mM Tris [pH 7.5], 150 mM NaCl) that contained 1% bovine serum albumin and 1% skim milk. Tissue sections were incubated overnight at 4°C with a rabbit polyclonal antibody against mouse LRP8 [25] diluted to 1:300 in TBS that contained 1% normal cow serum. Negative control tissue sections were incubated similarly with or without normal rabbit serum. After three 5-min washes in TBS, primary antibody-LRP8 complexes were detected by incubation for 2 h at room temperature with alkaline phosphatase-conjugated monoclonal anti-rabbit IgG (Sigma) diluted 1:200 in TBS blocking buffer. Tissue sections were washed three times in TBS, and incubated with the NBT/BCIP alkaline phosphatase substrate (Roche Applied Science). Sections were mounted in 5% gelatin, 27% glycerol, and 0.1% sodium azide. Photographs were taken under bright-field illumination using a Nikon Eclipse E800 microscope equipped with a digital camera (Nikon DXM 1200). Digital images were processed using the Photoshop software (Adobe Systems Inc., San Jose, CA) and assembled with the Illustrator software (Adobe Systems).
Gene-specific (LRP8, LDLR, VLDLR, RELN, MAPK8IP1, MAPK8IP2, DAB1) signals were normalized with the corresponding GAPD signals for each sample. Homogeneity of variance between follicular group and CL was verified by the O'Brien and Brown-Forsythe tests. Corrected values of gene-specific mRNA levels were compared between the follicular and CL groups by one-way ANOVA. When ANOVA indicated significant differences (P < 0.05), multiple comparisons of individual means for the SF, DF, OF and CL groups were compared by the Tukey-Kramer test (P < 0.05). The Dunnett test (P < 0.05) was used to compare individual means for the temporal hCG-induced follicular wall samples. Statistical analyses were performed using the JMP software (SAS Institute, Cary, NC). Data are presented as means ± SEM.
Cloning and Characterization of Bovine LRP8 cDNA
Three strategies were used to clone the bovine LRP8 cDNA. First, a 500-bp bovine LRP8 cDNA fragment, which was generated in a previous mRNA gene profiling experiment [6], was used as a probe to screen by hybridization a size-selected (between 3.5 kb and 4.5 kb) cDNA library, which was generated from bovine GC that were collected from DFs at D5 of the estrus cycle. The sequencing results showed that the longest cDNA fragment isolated did not contain the full-length ORF and started at G163 (Fig. 1). The 5'-RACE method was used as an alternative cloning approach and yielded only part of the missing 5'-end of the LRP8 cDNA despite numerous attempts. The third cloning approach consisted of screening a bovine genomic library with the most 5'-upstream cDNA fragment of the bovine LRP8 cDNA. Positive hybridizing clones were isolated and shown to contain the complete 5'-end of the bovine LRP8 ORF as well as the 5'-flanking DNA. Collectively, these results reveal that the bovine LRP8 cDNA cloned from GC consists of 4535 bp, and this nucleotide sequence was submitted to GenBank (accession number AY364441). This cDNA is composed of a 5'-UTR of 552 bp, an ORF of 2490 bp (including the stop codon), and a 3'-UTR of 1493 bp that contains seven copies of an AU-rich element (ATTTA), a motif that is known to contribute to short-lived mRNA [27], as well as a polyadenylation signal (AATAAA) followed by a poly(A)+ tail. The nucleic acid sequence in the vicinity of the start codon (300 to 600 bp; AY364441) is particularly rich in guanosine and cytosine residues (84%). The latter is known to promote the formation of strong secondary structures in mRNA that impede the ability of reverse transcriptase to generate full-length cDNA from mRNA, and contribute to resistance against DNA strand dissociation during the PCR reaction used in 5'-RACE cDNA cloning procedures.
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The ORF of the bovine LRP8 cDNA codes for an 829-amino acid protein, with a theoretical molecular mass (Mr) of 91 500 and an isoelectric point (pI) of 4.9 (Fig. 1). Amino acid homology searching in GenBank by PsiBLAST revealed orthologous proteins, with an overall identity of 87% and 85% for the human (NM_033300) and mouse (AJ312058) proteins, respectively. The characterized bovine LRP8 protein is 36 amino acids longer than the human LRP8 variant 2 (NM_033300) [13] and 41 amino acids shorter than the mouse protein (AJ312058) [28] (Fig. 1). The bovine LRP8 protein is structurally composed of a long extracellular amino-terminal ligand-binding segment (M1 to A691), a single transmembrane segment (A692 to W714), and an intracellular segment (R715 to P829). The extracellular ligand-binding segment contains a signal peptide (M1 to A27) that should be cleaved after A27, and conserved domains, such as four copies of a cysteine-rich LDL receptor A domain repeat (LDLR-A; K40-C76; K79-C117; Q120-C158; G163-C199), an EGF-like domain (C206-C240), an EGF-calcium domain (D242-C280), five copies of a LDL receptor B domain repeat (LDLR-B; N328-V373; K375-L416; G418-L460; Q462-D505; K506-E546), an O-linked sugar domain (A603-Y678), and four potential N-glycosylation sites (N384, N404, N638, N637) (Fig. 1). The cytoplasmic segment contains the consensus tetrapeptide 729NPVY732 sequence as an internalization signal [13, 29], a 59-amino acid (A759-K817) insert [14], and two potential consensus cAMP/cGMP-dependent protein kinase phosphorylation sites (T722, T736).
Tissue Distributions of Bovine LRP8 mRNA and Splicing Variants in Granulosa Cells
The expression patterns of bovine LRP8 mRNA in various bovine tissues were compared by RT-PCR/Southern blot analysis. The results revealed high levels of LRP8 mRNA in the GC from DFs collected at D5 of the estrus cycle and in GC from follicles collected on the third day following initiation of an ovarian stimulation treatment. Conversely, LRP8 mRNA expression was not detected in the other tissues analyzed (Fig. 2).
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The expression of LRP8 mRNA variants was studied in the GC of DFs at D5 of the estrus cycle, since LRP8 transcript variants have been reported in human, mouse, and chicken tissues. A bovine LRP8 cDNA that lacks the putative O-linked sugar domain was first cloned by screening the bovine GC size-selected cDNA library. Thus, we investigated the possible expression of a bovine LRP8 variant that contains this domain by RT-PCR using total RNA extracted from GC and oligonucleotide primers that flank the putative O-linked sugar domain (A603-Y678; Fig. 1). The RT-PCR generated major (487-bp) and minor (259-bp) cDNA products (Fig. 3A, lane 1). The PCR products were purified and characterized by sequencing, and were found to correspond to transcript variants that contained or lacked the putative O-linked sugar domain. These results indicate that the major form of the LRP8 receptor expressed in GC contains the putative O-linked sugar domain.
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The most notable difference between LRP8 and the other closely related members of the LDL-receptor gene family is the presence of a 59-amino acid (A759-K817; Fig. 1) residue insert in the cytoplasmic tail that is encoded by an additional exon. Since we first isolated a bovine cDNA that lacks this insert, we wished to determine whether a bovine variant that contains this sequence is expressed in the GC of DFs. Using primers that flanked the relevant region, RT-PCR analysis generated two cDNA products of 555 bp and 378 bp, which were represented at a similar level of expression (Fig. 3A, lane 2) and corresponded to transcript variants that contained or lacked the 59-amino acid stretch.
Since the full-length human LRP8 receptor is composed of a long extracellular domain that is encoded by 16 exons [14], we investigated the possible expression of LRP8 receptor transcript that lacked exons within the extracellular domain. RT-PCR analyses were carried out using total RNA from GC, and primers anchored in either the first, second or third exon encoding the extracellular amino-terminal of the receptor, in combination with a second primer anchored in exon 7. The RT-PCR generated a cDNA product for each pair of primers (Fig. 3B) of 523 bp, 404 bp or 307 bp. Characterization of these fragments by sequencing revealed that they all lack exon 5, which encodes for 129 amino acids that should be located between S161 and L162 of the bovine sequence when compared to the human LRP8 variant 1 protein (D50678) [14].
Regulation of LRP8, LDLR, and VLDLR mRNAs in Granulosa Cells
The expression of the LRP8 A759-K817 mRNA variant that harbors the 59-amino acid cytoplasmic insert was analyzed by semiquantitative RT-PCR in GC samples collected from follicles obtained at different developmental stages, including SFs (2–4 mm), DFs at D5 of the estrus cycle, OFs collected 24 h after injection of an ovulatory dose of hCG, and corpus luteum (CL) at D5. The semiquantitative RT-PCR demonstrated highest expression in the GC of DFs compared to other follicles and CL (P < 0.0001) (Fig. 4). The expression of LRP8 mRNA was 3.4-fold higher in DFs than in SFs. Since LRP8 shares high levels of homology with LDLR and VLDLR and the expression of these receptors has not been described in bovine GC during follicular development in a synchronized estrus cycle and CL, the expression levels of their respective mRNAs were compared by semiquantitative RT-PCR. The results show that while the LDLR and VLDLR mRNAs were expressed in all the GC extracts and CL, there were no significant variations in their respective mRNA levels (Fig. 4).
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Since hCG treatment caused downregulation of LRP8 mRNA in GC 24 h after its injection, semiquantitative RT-PCR was used to study the expression of the LRP8 A759-K817 mRNA variant in follicular walls obtained from ovulatory follicles that were isolated at different time-points between 0 and 24 h after hCG treatment (Fig. 5). The levels of LRP8 mRNA were elevated prior to hCG treatment, but decreased by 3.2-fold 12 h after hCG injection, and reached almost undetectable levels after 24 h (P < 0.0001).
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Regulation and Localization of LRP8 Protein in Ovarian Follicles
To determine whether the increase in LRP8 mRNA in bovine DFs was associated with changes in the protein levels, a specific antibody raised against the entire intracellular segment of mouse LRP8 was used in immunoblot analyses of GC protein extracts from follicles at different developmental stages. Three immunoreactive signals were detected in all the GC extracts studied: a 145 x 103 Mr band, which is believed to correspond to the full-length protein, and a double protein band of between 95 and 105 x 103 Mr, which is thought to represent splicing variants and/or the nonglycosylated precursor of LRP8 (Fig. 6). A significant increase in all the LRP8 protein isoforms was observed in the GC extracts of D5 DFs compared to SFs (P < 0.05), mirroring what was observed for LRP8 mRNA. Furthermore, the LRP8 signals decreased significantly with time in the GC extracts and reached the lowest level 24 h after hCG injection (P < 0.05), in agreement with the mRNA analyses. Conversely, LRP8 protein was not detected in the CL (Fig. 6).
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To determine the cellular localization of LRP8 protein expression in the bovine ovary, immunohistochemistry was performed using the antibody employed for immunoblotting, to probe sections of follicles obtained at different developmental stages, as well as the CL. Intense staining for LRP8 proteins was detected in the GC layer of DFs, whereas the theca layer and stromal tissue were not immunolabeled (Fig. 7, A and B). Immunostaining was mainly located at the cytoplasmic membrane of the GC (Fig. 7B). A marked decrease in LRP8 immunoreactivity was observed in the GC layer of OFs isolated 24 h after hCG treatment (Fig. 7, C and D). Preantral follicles at different developmental stages were negative for immunostaining, whereas small antral follicles (2–4 mm) showed faint to absent immunostaining (Fig. 7E). No labeling was observed in follicles in late atresia (not shown) or in luteal cells (Fig. 7F).
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Expression of RELN, MAPK8IP1, MAPK8IP2, and DAB1 mRNAs in Periovulatory Follicles
In nervous system tissues, RELN is the natural ligand for LRP8, whereas MAPK8IP1, MAPK8IP2, and DAB1 have been shown to interact with the intracellular segment of LRP8 following RELN binding [18]. Since LRP8 was found to be differentially expressed in the GC of bovine DFs, we wished to determine by semiquantitative RT-PCR whether RELN, MAPK8IP1, MAPK8IP2, and DAB1 mRNAs were coexpressed in the DFs. The results demonstrate that RELN expression is maximal in the TC of DFs, is decreased significantly in the TC of OFs (P < 0.0037; Fig. 8), and is absent in the GC and CL (data not shown). MAPK8IP1 mRNA was shown to be expressed in the GC of DFs and increased in OFs (P < 0.037; Fig. 8). Conversely, MAPK8IP2 and DAB1 were not detected in the GC (data not shown).
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This study is the first to document the cloning and characterization of full-length bovine LRP8 cDNA, and to demonstrate the expression of several isoforms of LRP8 in the GC of bovine DFs using an in vivo model. Furthermore, we report the differential expression of RELN mRNA, an LRP8 ligand, in the thecal layer of DFs, whereas MAPK8IP1 mRNA, a LRP8 scaffolding protein that is involved in intracellular LRP8 signaling, is expressed in GC. These observations suggest the existence of a new interaction mechanism between the thecal and granulosa layers through RELN and LRP8/MAPK8IP1 signaling in the DF.
Our comparative analysis reveals that the amino acid sequence of the full-length bovine LRP8 protein is similar to those of the human and mouse proteins, and that all of the known structural and functional domains are conserved. Several splicing variants in the extracellular segment of LRP8 are expressed in the GC of DFs, although they all lack ligand-binding repeats 4–6 (encoded by exon 5) as compared to the human protein [14]. Differentially spliced receptors with three, four or five repeats are also expressed in human, mouse, and chicken [14, 15, 28, 29]. Human LRP8 splice variants that harbor three or seven repeats have identical affinities for ß-VLDL [14, 30]. Therefore, the functional significance of the different splice variants in the extracellular segment of LRP8 remains unknown. LRP8 can also be differentially spliced in the cytoplasmic segment, to generate a longer version that contains a unique 59-amino acid proline-rich insert (A759-K817) that is encoded by exon 18, as characterized in humans and mice [15]. In the present study, we demonstrate that a bovine variant that harbors this insert is expressed in the GC of DFs at a level similar to that of the short isoform, and its amino acid composition is highly conserved with its mammalian orthologs.
Previous studies have reported higher expression of LRP8 mRNA in bovine GC of DFs compared to SFs (2–4 mm), and at the time of DF selection, LRP8 mRNA was most abundant in the future DF compared to the largest subordinate follicle [6, 8]. Our RT-PCR and immunoblotting results reveal that LRP8 mRNA and proteins are specifically expressed in GC, with almost exclusive expression in the DF, with faint to undetectable labeling in small antral follicles (2–4 mm), and no expression in preantral or atretic follicles. However, no protein expression was detected in the GC of small antral follicles by immunohistochemistry. This discrepancy may be explained by the pooling of GC from 20 SFs (2–4 mm) per group at different stages of development, which were obtained from ovaries at various stages of the estrus cycle for the RT-PCR and immunoblotting studies, whereas individual 2–4-mm follicles were observed by immunohistochemistry. The pooling of GC from SFs was necessary to amass sufficient amounts of mRNA and protein for the analyses. A previous study using RT-PCR analysis has reported that LRP8 mRNA is expressed in bovine TC [31]. However, in the latter study, the preparations of TC were not evaluated for possible contamination with GC, and the reverse primer used in their RT-PCR to evaluate LRP8 expression does not match the described bovine LRP8 sequence (AY364441). It is well known that TC are embedded in ovarian connective tissues and are often contaminated by GC [32, 33]. The TC preparations used in the present study were analyzed by RT-PCR for the presence of CYP19A1 mRNA, which is a specific GC marker, and the results show that the TC samples collected from the DFs were slightly contaminated by GC (data not shown). Conversely, our GC preparations were not contaminated by TC, as confirmed by the absence of cytochrome P450, family 17, subfamily A, and polypeptide 1 (CYP17A1) mRNA expression (data not shown). Based on our immunohistochemical results, we conclude that LRP8 is not expressed in bovine TC.
The pattern of LRP8 expression correlates with the capacity of follicles to synthesize high levels of steroids. In fact, during the recruitment phase of follicular development, the CYP19A1 mRNA levels increase in the follicles [34]. After selection, the DF expresses higher levels of mRNAs for LHCGR and enzymes that are required for progestin and androgen synthesis, such as hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase (HSD3B2), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1), steroidogenic acute regulator (StAR), and CYP17A1, than the unrecruited follicles [6, 7, 34]. Moreover, the concentrations of apolipoproteins, such as LDL and VLDL, increase in the follicular fluids of medium- and large-sized antral follicles (4–10 mm) compared to small antral follicles (2–4 mm) [35]. Collectively, these reported observations and our present data suggest that LRP8 contributes to cholesterol uptake in GC during follicular growth and dominance, at which time the cholesterol requirement for steroidogenic activity is increased.
Cholesterol transport protein apolipoprotein E (APOE), which associates with cholesterol-rich lipoproteins, such as LDL and VLDL, and enables binding to members of the LDL-receptor family, is expressed in the theca/interstitial cells (TIC) of rodents [36]. Furthermore, APOE interacts with unknown members of the LDL-receptor family to increase transcription of the enzyme CYP17A1 and stimulate TIC androgen production [37]. We demonstrate that LRP8 is not present in bovine TC that are known to express CYP17A1, which excludes LRP8 as the LDL-receptor involved in the regulation of androgen synthesis by CYP17A1 in the bovine ovary. Our data further suggest that LRP8 has functions other than cholesterol uptake during follicular growth, since LRP8 mRNA and protein are downregulated during the ovulatory process and are absent in the CL. The downregulation or absence of LRP8 contrasts markedly with the increased cholesterol requirements during ovulation and luteinization [38, 39]. In contrast to LRP8, we show that LDLR and VLDLR, which are already known to mediate cholesterol uptake in the ovary [31, 35, 40], are expressed in bovine GC of OFs 24 h post-hCG and in the CL.
Besides its role as a classical endocytotic receptor, LRP8 regulates multiple intracellular signaling cascades. LRP8 and VLDLR are components of a signaling pathway that relays the RELN signal into neurons for proper neuronal positioning during brain development, and for learning and memory in the adult brain [16–18, 41]. RELN interacts with the extracellular domains of LRP8 and VLDLR, causes receptor clustering [42], and triggers activation of intracellular signaling pathways [43]. One intracellular interacting partner of LRP8 and VLDLR is DAB1. Upon binding of RELN to LRP8 or VLDLR, DAB1 is phosphorylated and activates pathways that control cell motility, adhesion, and shape [43–47]. In the mouse ovary, RELN mRNA is expressed in the interstitial region but not in the follicles [48]. Our data show that bovine RELN mRNA is mainly expressed in TC preparations of DFs, downregulated in TC preparations of OFs 24 h post-hCG, and absent in GC and luteal cells. Therefore, RELN mRNA in the theca layer and LRP8 mRNA and protein in the GC are predominantly expressed in the DF, and are similarly downregulated during the ovulatory process and luteinization. In addition, we performed an RT-PCR analysis on the GC of DFs to determine whether DAB1 mRNA is expressed in the DF. The levels of DAB1 mRNA were low to undetectable, and further studies are needed in order to investigate whether DAB1 protein is present in the follicle and participates in mediating LRP8 and/or VLDLR signaling in GC.
Intracellular proteins that interact specifically with the 59-amino acid insert (A759-K817) of LRP8 include MAPK8IP1 and MAPK8IP2 [12]. Our data demonstrate that MAPK8IP1 and LRP8 mRNAs are expressed in the GC of the DF, which suggests that these proteins also interact in GC. MAPK8IP1 has been shown to scaffold different proteins that regulate the c-Jun N-terminal kinase (JNK, MAPK8) [11, 12] and AKT1 [49] signaling pathways. Inhibition of JNK [50] or activation of AKT1 [49] by MAPK8IP1 reduces apoptosis. MAPK8IP1 also interacts with RhoGEF, the exchange factor for the small GTPase rhoA, which is involved in the rearrangement of the actin cytoskeleton [51]. We hypothesize that MAPK8IP1 participates in preventing apoptosis or modulates cellular activity by acting on cytoskeletal components in the GC of the DF. We also report that MAPK8IP1 mRNA expression increases in the GC of hCG-induced OFs at a time when LRP8 expression is downregulated. It is possible that MAPK8IP1 regulates JNK activation to prevent apoptosis during cellular stress induced by ovulation using a pathway that does not involve LRP8 [52]. Moreover, in hCG-induced OFs, MAPK8IP1 may interact with JNK, which is proposed to activate RHOX5, a transcription factor that is expressed in the mural GC of periovulatory follicles [53].
ACKNOWLEDGMENTS
The authors thank Manon Salvas for technical assistance during the nucleic acid sequencing, and Dr. Christine Theoret (Université de Montréal) for constructive comments on the manuscript.
FOOTNOTES
1Supported by a Discovery Grant to J.G.L. from the Natural Sciences and Engineering Research Council of Canada (NSERC), and a fellowship to T.F. from the Fonds Québecois de la Recherche sur la Nature et les Technologies (FQRNT). This work was presented in part at the 38th Annual Meeting of the Society for the Study of Reproduction, July 24–27, 2005, Quebec City, Quebec, Canada. The nucleotide sequence reported in this paper has been submitted to GenBank under the accession number AY364441. 
Correspondence: 2Jacques G. Lussier, Faculté de Médecine Vétérinaire, Université de Montréal, P.O. Box 5000, St-Hyacinthe, Québec, Canada J2S 7C6. FAX: 450 778 8103; e-mail: jacques.lussier{at}umontreal.ca
Received: 12 September 2006.
First decision: 4 October 2006.
Accepted: 9 November 2006.
REFERENCES
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8-mm follicles that were collected on D3 of a 4-day gonadotropin ovarian stimulation protocol (SOV).
