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Ovary |
Institute of Medical Biochemistry, Department of Molecular Genetics, BioCenter and University of Vienna, A-1030 Vienna, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, granulosa cells, oocyte development, ovary, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Given the high degree of specialization of oocytes in oviparous species for efficient endocytosis and the effects of mutant dynamin on both synaptogenesis and oocyte endocytosis [2], we became interested in endophilins, because they, too, are involved in clathrin-mediated endocytosis as well as in synaptogenesis [15–18]. The type A endophilins (here termed endophilin I, II, or III) are presynaptic proteins that bind to the GTPase dynamin and the polyphosphoinositide-phosphatase synaptojanin that are implicated in endocytosis and vesicle recycling. A likely role of the endophilins in the membrane remodeling required for vesicle formation could result from the lysophosphatidic acid acyltransferase activity of endophilin I [19], but the direct interaction of endophilin with lipid bilayers per se also leads to tubular membranes [20, 21]. Considerably less is known about the functions of the other family members, endophilin II and endophilin III, or about the roles of endophilins beyond vesicle endocytosis (for review, see [21]), let alone in nonneuronal cell systems.
Thus, based on the lack of knowledge about the significance of the expression of different endophilin family members and on our interest in the biology of oocyte growth and follicle development, we performed a detailed molecular characterization of chicken endophilin I, II, and III. In addition to investigations at the biochemical and cell biological level, we utilized a naturally occurring mutation in the gene for the major yolk precursor receptor, which is functionally absent in nonlaying mutant "restricted ovulator" (R/O) hens [22–24], as a further tool in studying the roles of endophilins in oocyte-specific processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The R/O breeding colony, derived from a White Leghorn carrier [23, 25], was reared from fertilized eggs kindly provided by Dr. R.G. Elkin (Department of Poultry Science, Pennsylvania State University, State College, PA). Animals were maintained on layer's mash, with a 12L:12D photoperiod and free access to feed and water. The genotype of the animals at the lr8 locus was determined according to the method of Bujo et al. [25]. For the present experiments, we used adult normal laying hens and R/O hens of the colony. All procedures were performed according to the protocols approved by the Animal Care Committee of the University of Vienna.
Cloning of Chicken Endophilin I, II, and III
A polymerase chain reaction (PCR) fragment was obtained by reverse transcriptase-PCR (Life Technologies, Inc., Lofer, Austria) using chicken brain poly(A)+-RNA for first-strand cDNA synthesis and two synthetic oligonucleotides, 5'-AGCCACTCAGAAAGTGAGTGAGAA-3' and 5'-ATCACAGCCCTGCTGGTGACATCC-3', which correspond to a conserved region of human and mouse endophilin I cDNA (nucleotides 45–153 in human, nucleotides 120–227 in mouse). The PCR fragment was subcloned, and its sequence was found to be 92% identical to that of both human and mouse endophilin I. The same procedures were applied toward the cloning of chicken endophilin II and III. The 5'- and 3'-ends of chicken endophilin mRNAs were cloned with the rapid amplification of cDNA ends (5'/3'-RACE kit; Roche, Vienna, Austria) using an oligonucleotide from the known part of the endophilin sequence and an anchor oligonucleotide. The PCR products were subcloned into the pCR 2.1 TOPO vector (Invitrogen, Lofer, Austria), and their sequences were determined on an ABI-sequencer.
Northern Blot Analysis
Tissues were dissected immediately after the animals were killed. The terms ovary (see Fig. 3) and stroma refer to the tissue after removal of all adherent follicles of a diameter larger than 1 mm. From the oviduct, we used the magnum portion (see Fig. 3). Total RNA prepared from various tissues of adult male and female chickens was denatured using glyoxal and dimethyl sulfoxide, separated by electrophoresis on a 1.0% agarose gel, and blotted onto Hybond N+ nylon membrane (Amersham Pharmacia Biotech, Vienna, Austria) using standard methods. As probes, we used 432-, 974-, and 740-base pair PCR products corresponding to chicken endophilin I, II, and III cDNA fragments, respectively, and labeled with 32P using the Megaprime DNA labeling kit (Amersham Pharmacia Biotech). The membrane was hybridized overnight at 65°C in a solution containing 10 mg/ml of bovine serum albumin, 70 mg/ml of SDS, 0.5 M sodium phosphate buffer (pH 6.8), 1 mM EDTA, and the 32P-labeled DNA probe. Washing was performed at 65°C in 5 mg/ml of bovine serum albumin, 50 mg/ml of SDS, 40 mM sodium phosphate buffer (pH 6.8), and 1 mM EDTA and then in 10 mg/ml of SDS, 40 mM sodium phosphate buffer (pH 6.8), and 1 mM EDTA. The Hybond filter was exposed to Reflection film (NEN Life Science Products, Vienna, Austria) with intensifying screens at -80°C. The relative amounts of RNA loaded were estimated using methylene blue staining of ribosomal RNA.
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Production of Fusion Proteins and of Anti-Chicken Endophilin Antibodies
Constructs of glutathione-S-transferase-[GST] fusion proteins were used for immunization and pull-down experiments (see Fig. 5B). The indicated partial cDNA fragments of endophilins were PCR-amplified using Pfu DNA polymerase (Stratagene, Amsterdam, The Netherlands) and specific 5'- and 3'-end chicken endophilin oligonucleotides and were subcloned via blunt-end ligation into pCR-Script SK(+) (Stratagene). The identity of the PCR-generated fragments was verified by sequencing. The obtained partial cDNA fragments were then directionally cloned in-frame into a pGEX-5X expression vector (Amersham Pharmacia Biotech), followed by transfection of Escherichia coli XL1 Blue subcloning-grade competent cells (Stratagene). The GST-fusion protein was induced by incubating the cells in the presence of 0.1 mM isopropylthio-D-galactoside and purified according to the method described by Frangioni and Neel [26].
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Antisera against the chicken endophilins were prepared using GST-fusion proteins containing chicken endophilin fragments corresponding to nonconserved regions with or without the SH3 domain. Specifically, the fragment of chicken endophilin I consisted of residues 234–353, that of chicken endophilin II of residues 234–308 (lacking the SH3 domain), and that of chicken endophilin III of residues 216–353 of the proteins (see Fig. 5B). For pull-down assays, GST-fusion proteins containing the SH3 domains of chicken endophilin I, II, or III (see Fig. 5B); endophilin III lacking the SH3 domain; or GST alone were prepared. The GST-fusion proteins were purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and were used for immunization of adult female New Zealand White rabbits. For the first injection (Day 0), the antigens were mixed with Freund complete adjuvant, and for successive booster injections thereafter (Days 14, 21, and 28), the antigens were mixed with Freund incomplete adjuvant. Antisera were collected after Day 35 and every 2 wk thereafter for a period of 2 mo. Nonimmune serum was obtained before immunization.
Preparation of Triton X-100 Extracts
Freshly obtained samples were placed in ice-cold homogenization buffer (4 ml/g wet wt) containing 20 mM Hepes (pH 7.4), 300 mM sucrose, 150 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, and 2.5 µM leupeptin and were then homogenized with an Ultra-Turrax T25 homogenizer (VWR, Vienna, Austria) (7-mm probe) three times for 20 sec each time. Total homogenates were spun at 620 x g for 10 min at 4°C, and 1/20 volume of 20% Triton X-100 was added to the supernatant. After a 30-min incubation at 4°C, samples were centrifuged at 300 000 x g for 60 min at 4°C. The resulting supernatant was stored in aliquots at -20°C until use. Protein concentrations were determined by the Bio-Rad Protein Assay (Bio-Rad, Vienna, Austria). For preparation of Triton X-100 extracts of chicken ovarian follicles, the follicles (diameter, 2–20 mm) were first punctured, and the yolk was then carefully squeezed out, followed by a 30-sec wash in PBS (pH 7.4). The remaining follicle tissue was then processed as described above.
SDS-PAGE and Immunoblotting
One-dimensional 12% SDS-PAGE was performed using a minigel system (Mini-Protean II; Bio-Rad). Samples were prepared in the presence (reducing conditions) or absence (nonreducing conditions) of 10 mM dithiothreitol. Electrophoresis was performed at 180 V for 1 h with the inclusion of broad-range Mr standards (Bio-Rad). Electrophoretic transfer of the proteins to nitrocellulose membrane (Hybond-C; Amersham Pharmacia Biotech) was performed in transfer buffer (26 mM Tris, 192 mM glycine, and 20% methanol) for 1 h at 200 mA, on ice, using the Mini Transblot system (Bio-Rad). The transfer was verified by staining the nitrocellulose membrane with 0.2% Ponceau S in 3% (w/v) trichloroacetic acid and then destaining in water. Western blot analysis was performed using specific rabbit antisera at the concentrations indicated in the figure legends, followed by horseradish peroxidase-labeled protein A (1:5000; Sigma, Vienna, Austria) and the chemiluminescence detection method (ECL system; Amersham Pharmacia Biotech). The polyclonal rabbit anti-chicken clusterin antibody directed against the carboxyterminal subunit has been described previously (antibody B in Mahon et al. [27]).
Immunohistochemistry
Ovarian follicles (yolk-less "white"; diameter, <4 mm) from normal or R/O hens and ovarian stroma (containing oocytes with a diameter of 60–500 µm) were treated overnight with 5% and 30% sucrose at 23°C. Following three successive washes with PBS, the follicles were transferred to plastic moulds. Cryostat sections (thickness, 18 µm) were obtained on a Microtome (HM 500 OM; MICROM GmbH, Walldorf, Germany), transferred onto glass slides, fixed for 5 min with ice-cold acetone:methanol (1:1), and then washed for 5 min with PBS. For immunohistochemistry, the tissue sections were pretreated with 10% H2O2 for 10 min. The sections were incubated in PBS containing 1% nonfat powdered milk and 3% goat serum (blocking buffer) for 1 h at room temperature. After five washes with PBS for 1 min each wash, the sections were incubated for 1 h at room temperature in a humid chamber in blocking buffer containing primary antisera against chicken clusterin; endophilin I, II, or III; or corresponding preimmune sera at the concentrations indicated in the figure legends. After being washed five times with PBS, the follicle sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (1:500) in blocking buffer for 1 h at 23°C. Slides were subsequently washed five times in PBS and incubated with avidin-horseradish peroxidase (1:200) in blocking buffer without goat serum for 1 h at 23°C. Following extensive washing in PBS, the sections were incubated in 0.1 M sodium acetate buffer (pH 5.2) containing 0.03% H2O2 and 0.2 mg/ml of 3-amino-9-ethylcarbazole. The color reaction was followed under a microscope and terminated by incubating the slides in water. The stained sections were mounted in Aquamount (BDH, Wesel, Germany), and photographs were taken with a Zeiss Axiovert 10 light microscope (Jena, Germany).
Pull-Down Assays
Chicken and rat brain extracts (200 µg of protein) were prepared as described above and incubated overnight at 4°C with 25 µg of the GST-fusion proteins of chicken endophilin I, II, or III containing the SH3 domain (see Fig. 5B); endophilin III lacking the SH3 domain; or GST alone prebound to glutathione-Sepharose beads. The beads were subsequently washed three times with PBS, eluted with electrophoresis sample buffer, and prepared for SDS-PAGE. The gels were stained by Gel Code Blue stain reagent (Pierce, Vienna, Austria) or used for Western blot analysis with anti-rat dynamin antibody at 1:4000 dilution or anti-rat synaptojanin antibody at 1:1000 dilution.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The isolated full-length cDNAs for chicken endophilin I, II, and III revealed open reading frames encoding proteins of 353, 367, and 353 amino acids, respectively (GenBank/EMBL Data Bank accession nos. AJ439350—AJ439352). The chicken endophilin polypeptides are approximately 70% identical among themselves, with the SH3 domains particularly conserved at 86%–90% identity (Fig. 1). The predicted mature chicken endophilin I polypeptide (353 residues) has a calculated Mr of 39.9 kDa and shows 90%, 90%, and 91% identity to the human, mouse, and rat homologues, respectively (Fig. 2). The predicted mature chicken endophilin II protein (367 residues, calculated Mr of 41.7 kDa) is also approximately 90% identical to human, mouse, and rat endophilin II (Fig. 2). Chicken endophilin III cDNA shows 78% and 76% identity to human and mouse endophilin III cDNA, respectively (data not shown). The predicted mature chicken endophilin III polypeptide (353 residues, calculated Mr of 40 kDa) shows 80%–82% identity with the human, mouse, and rat proteins (Fig. 2). Furthermore, the glycine and proline residues in the SH3 domain, the consensus region for protein-protein interaction in GRB2 [28, 29], are identical in all chicken endophilins (G-F-F-P) (Fig. 1, asterisks) and are totally conserved in the SH3 domains of chicken amphiphysin I (G-L-F-P) and chicken FAP52 /pacsin II/syndapin II (G-L-Y-P) [30, 31].
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Tissue Distribution and Expression of Chicken Endophilins
With the sequences in hand, we determined the expression patterns of the individual chicken endophilins at the transcript level. Northern blot analysis of total RNA from the examined tissues revealed that the three endophilins showed different expression profiles (Fig. 3). A single, approximately 3.0-kilobase (kb) endophilin I transcript was present at high levels only in brain. The main endophilin II transcript (
3.8 kb) was present in most tissues, with high levels in brain and small ovarian follicles. An additional transcript (>4 kb) was seen in brain (Fig. 3) and in the chicken embryonic fibroblast cell line, Cef 32 (data not shown), raising the possibility that one or more closely related endophilin transcripts may exist in the chicken. Finally, a single, approximately 2.5-kb endophilin III transcript was present at high levels in brain, testis, and small follicles and at lower levels in adrenal glands. These data are comparable to those in the rat, mouse, and human as far as they have been reported, and they also show very similar distribution to that of mammalian dynamin I, II, and III [32, 33]. Particularly intriguing are the finding that in the bird, as in mammals, endophilins are undetectable in the liver and the discovery of significant expression of endophilin II and III in ovarian follicles.
Endophilin II and III Expression During Ovarian Follicle Development
Triggered by the finding that significant levels of endophilin II and III are present in the follicles and based on our interest in the biology of oocyte growth and follicle development, it was important to establish the pattern of endophilin expression in ovarian follicles in greater detail (Fig. 4). If expression correlates with oocyte growth via endocytosis, then a role for endophilins in the process could be inferred. Thus, we analyzed follicles in different developmental phases (i.e., with different diameters). Figure 4A shows the results obtained with phase I follicles (termed white, because their oocytes lack yolk), either very small (diameter, 1 mm), small (diameter, 1–3 mm), or large (diameter, 3–5 mm), and phase II follicles (these contain oocytes that have begun to take up yolk), including small yellow (diameter, 5–6 mm) and large yellow (diameter, 6–9 mm). In Figure 4B, analysis of extraoocytic cells of phase III follicles, characterized by F5 (diameter, 10 mm) to F1 (diameter, >30 mm) follicles, with the latter being the next follicle to ovulate, is shown. Endophilin II transcript was detectable in whole follicles (Fig. 4A), whereas it was found in granulosa cells (Fig. 4B, GC5 through GC1) and theca cells (Fig. 4B, TH5 through TH1) of F5 to F1 follicles throughout the entire rapid growth period. Small follicles contained the highest levels of endophilin II, which decreased with increasing follicle size, when committed follicles grow rapidly by acquisition of yolk. Interestingly, high levels of endophilin II transcripts (but lack of endophilin III; not shown) were also apparent at the opposite end of follicle development, namely in the postovulatory sac (i.e., the tissue that remains following release of the F1 oocyte into the oviduct) (Fig. 4A, POV.sac.). On the other hand, endophilin III transcripts were detectable only during the early stages of follicles and then decreased rather sharply (Fig. 4A). A higher ratio of endophilin III to endophilin II transcript levels in small follicles, but low endophilin III levels in granulosa cells (Fig. 4B) and absence from theca cells (not shown), strongly suggested that endophilin III is preferentially expressed in the oocytes, at least up to the large-white stage. This situation appeared to be analogous to that previously observed for the main yolk receptor in oocytes, LR8 [24], a variant of which is expressed at low levels in granulosa cells [9]. Thus, we next analyzed directly the distribution of the endophilin II and III proteins in the different cell types of the follicle (i.e., oocyte, granulosa cells, and theca cells).
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Characterization of Antisera Against Chicken Endophilins and Analysis of Endophilin Protein Distribution in Avian Tissues and Cells
We first tested the specificity of our polyclonal antibodies. Immunoblotting analysis of the GST-fusion proteins revealed that each antibody specifically recognized its antigen (Fig. 5, A–C); in brain, which contains all three endophilins (see Fig. 3), each antiserum decorated a single band (Fig. 5C). Endophilin II has slower electrophoretic mobility than its homologues, which is compatible with its longer cDNA. With endophilin II, for example, we also showed good agreement between transcript levels (see Fig. 3) and protein expression as analyzed by immunoblotting (Fig. 5D). To obtain a detailed picture of the distribution of the individual endophilins in chicken follicles, we performed immunohistochemical analysis on phase I follicles (stroma or small white; diameter, <3 mm), which contain the highest levels of endophilin II and III transcripts (see Fig. 4) and are also the easiest to manipulate because of their low yolk content. Compatible with the results of the transcript studies, endophilin I was undetectable in follicles (Fig. 6D). Within the follicular structure, the most prominent site of endophilin II localization was in the cell layers in the central region of the theca (Fig. 6E); in contrast, the highest level of endophilin III protein (Fig. 6F) was found within the oocyte. Significantly, endophilin III in the oocyte displayed the same distribution as the chicken receptors LRP380 and LR8 (Fig. 6, F vs. H and I), two oocyte-specific endocytic receptors belonging to the LDL-receptor gene family (see Introduction). Further structural confirmation of the endocytically active compartment as the site of endophilin III localization in oocytes was facilitated by using our antibody to zona pellucida protein 1, a component of the extraoocytic fertilization coat [34]. Indeed, this antibody decorated the region at the border between oocyte and the granulosa cell layer (Fig. 6G, ZP), distinct from the region containing LR8, LRP380, and the endophilins.
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To obtain further evidence for involvement of endophilin III in oocytic endocytosis, we investigated its expression in tissues of a mutant chicken strain (i.e., the R/O strain [24]) in which oocytes fail to endocytose yolk precursors as a consequence of disruption of LR8 function by a point mutation in the lr8 gene [22, 23]. Up to a diameter of approximately 4 mm, when yolk incorporation commences, ovarian follicles of R/O hens are anatomically indistinguishable from those of normal ones. Figure 7 shows that such follicles from an R/O hen contain less endophilin III than those from a normal hen, whereas in the brains of normal and R/O hens, the levels are identical. We also determined the expression of clusterin, which we have previously shown to be increased in oocytes destined for follicular atresia [27]. Because the two types of follicles contain identical amounts of clusterin (Fig. 7, lanes 5 and 6), the decrease of endophilin III in the oocytes of the mutant animal (Fig. 7, lanes 1 and 2) is unlikely to result from a general apoptotic signal in R/O oocytes but, rather, is related to the oocyte's decreased endocytic capacity because of the receptor's inactivity.
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Immunohistochemical inspection of a follicle section from an R/O hen in comparison to a normal follicle (Fig. 8) revealed reduced amounts of endophilin III in R/O oocytes, showed unaltered levels or localization of endophilin II in theca cells, and confirmed the presence of clusterin in the granulosa cells of the two follicle types. Taken together, these data are compatible with endophilin III being an important component of the endocytic machinery of the germ cell that assures yolk uptake and oocyte growth. Endophilin II expression is less discriminant and, as indicated by its wide tissue distribution, is expected to play a role in endocytosis in various somatic cell types.
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Chicken Endophilins Are Functional: Protein-Protein Interactions
To obtain support for an involvement of chicken endophilins in oocytic receptor-mediated endocytosis, we tested whether the avian proteins display the required biochemical properties. One important function of mammalian endophilins is their interaction via the SH3 domains with the proline-rich domains of synaptojanin and dynamin [15, 17, 35, 36]. To demonstrate this function for chicken endophilins, we performed pull-down assays using GST-fusion proteins of chicken endophilin I, II, or III (Fig. 9) and brain extracts. With all GST-fusion proteins that contain the SH3 domains, two major proteins with apparent Mr of approximately 150 and 100 kDa, respectively, were pulled down from rat and chicken extracts (Fig. 9A, lanes 1–6). In contrast, pull-down with a GST-fusion protein representing endophilin III lacking the SH3 domain region (Fig. 9A, lane 7) or with GST-beads (Fig. 9A, lane 8) did not produce these bands. The sizes of the specifically coprecipitated proteins were compatible with those previously reported for rat synaptojanin and dynamin [15, 17, 35, 36], and indeed, Western blot analysis with anti-rat synaptojanin and anti-rat dynamin antibodies on aliquots of the pull-down precipitates confirmed their identities (Fig. 9B, lanes 1–6). These data show that the chicken endophilins characterized here share with their mammalian counterparts functional properties important for participation in clathrin-mediated endocytosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A shortcoming of current approaches to endophilin biology is the considerable lack of studies regarding these proteins in nonneuronal cell systems that display high constitutive endocytic activity. As a consequence, very little insight has been gained concerning the roles of endophilins and their binding partners in other cells that could provide valuable information at the molecular level. Therefore, to gain understanding of possibly more general functions of endophilins, we have begun to explore the growing chicken oocyte for investigation of clathrin-mediated processes and, in particular, of the endophilin family. The fact that in Drosophila sp. synaptogenesis as well as oocyte growth are disrupted by shibire, a mutation in dynamin I [39, 40], a major binding partner of endophilin, is an additional attractive feature of oocyte growth. We have previously shown that the major yolk receptor in chicken oocytes, LR8, constitutes 5% of the total amount of protein in the plasma membrane [41]. During the rapid phase of oocyte growth, as much as 1 g/day of protein is taken up into the germ cell, largely via LR8. In the mutant R/O strain, LR8 function is disrupted, and oocytes do not grow to term [22, 23].
Thus, because clathrin-mediated endocytosis is one of the major operating principles in oocyte growth, we postulated that it involves endophilin as an important component; this notion is indeed substantiated by the current findings as follows: First, these studies, to our knowledge for the first time, have characterized avian endophilins at the molecular level, revealing that their mammalian counterparts have been well conserved. Second, endophilins are expressed widely in chicken tissues and display typical functional properties, as demonstrated by their interaction with dynamin and synaptojanin. Third, within developing ovarian follicles, the different members of the endophilin family are expressed, albeit not exclusively, in different cell types, with endophilin III localized mainly to the endocytic compartment of oocytes. Fourth, in the yolk receptor-defective oocytes of R/O hens, endophilin III levels are significantly reduced, apparently coupled to their diminished clathrin-mediated endocytic activity.
A germ cell/somatic cell dichotomy in terms of expression of related genes in chicken gonads has been observed previously (for review, see [24]). Expression of different splice variants of LR8 in oocytes versus somatic cells in the ovary and in spermatids versus nongerm testicular cells [9, 10], as well as the expression of different pairs of LDL-receptor family genes in oocytes versus granulosa cells, point toward dichotomy as a general phenomenon. Of interest is the fact that the predominantly testicular endophilin III and the testis-type dynamin III are preferentially expressed in Sertoli cells, which elaborate endocytic processes and nurse developing germ cells, analogous to the role of granulosa cells for the oocyte. Furthermore, human endophilin III [42, 43] exists in splice variants that display different tissue specificities [44]. Targeting to different cellular compartments may allow variants of the endophilin and dynamin family to carry out specific functions. One example of this notion is a recent study on intersectin, a newly identified, SH3 domain-containing protein with affinity for dynamin, synaptojanin, and others (epsin, Eps 15, SNAP 25/23, and mSOS) [45, 46]; those results suggest that in neurons and nonneuronal cells, different splice variants of intersectin are components of the respective endocytic machinery.
At least in the chicken, the endophilin family members do not appear to be redundant, despite their common binding properties for dynamin and synaptojanin, because they are present in different tissues and at different developmental stages. Overlapping localization of endophilin I and II has been shown in synapses, with slightly different localization in central areas such as the hippocampal region [36]. As shown in the present study for ovarian follicles, chicken endophilin II and III are mainly expressed in theca cells and oocytes, respectively, which is compatible with roles of these SH3 proteins not only in neuronal but also in nonneuronal cells. The present data strengthen the notion that endophilins contribute significantly, likely in cell type-specific fashion, to general endocytic activities that are important for the key step in reproduction (i.e., follicle development).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Wolfgang Johann Schneider, Institute of Medical Biochemistry, Department of Molecular Genetics, BioCenter and University of Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria. FAX: 43 1 4277 61804; wjs{at}mol.univie.ac.at
Received: 17 October 2002.
First decision: 14 November 2002.
Accepted: 10 December 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin receptor family. Ann N Y Acad Sci 1994 737:145-153[Medline]This article has been cited by other articles:
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  S. Hummel, A. Osanger, T. M. Bajari, M. Balasubramani, W. Halfter, J. Nimpf, and W. J. Schneider Extracellular Matrices of the Avian Ovarian Follicle: MOLECULAR CHARACTERIZATION OF CHICKEN PERLECAN J. Biol. Chem., May 28, 2004; 279(22): 23486 - 23494. [Abstract] [Full Text] [PDF]
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SH3 in B), or III (lane 3; Endo III in B) were subjected to 12% SDS-PAGE under reducing conditions and processed for immunoblotting with the individual antisera (1:500 dilution) raised against the respective antigens as indicated. B) Schematic representation of the constructs for GST-endophilin fusion proteins used for immunization and/or pull-down assays (see Materials and Methods). The endophilin fragments used are indicated by the positions (amino acid numbers) of their respective amino- and carboxy-termini. C) Chicken brain extract (40 µg protein/lane) was subjected to 12% SDS-PAGE under reducing conditions and processed for immunoblotting with antisera raised against the individual endophilins as described in A. Numbers on the right correspond to the positions of migration of marker proteins (in kDa). D) Triton X-100 extracts (40 µg protein/lane) prepared from the indicated chicken tissues were analyzed by immunoblotting with serum raised against GST-endophilin II fusion protein (Endo II
