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ABSTRACT: At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.
Reproduction 08/2012; 144(4):423-31. · 2.58 Impact Factor
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ABSTRACT: Because of the presence of sperm-storage tubules (SST) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm have entered the female reproductive tract, they can survive for a prolonged time in domestic birds, although the specific mechanisms involved in the sperm uptake into, maintenance within, and controlled release from the SST remain to be elucidated. In this report, we provide evidence that progesterone triggers the release of the resident sperm from the SST in the UVJ. The ultrastructural observation of the SST indicated that the resident sperm are released from the SST around 20 h after oviposition. When laying birds were injected with progesterone, most of the sperm were released from the SST within 1 h of injection. In situ hybridization analyses demonstrated the presence of the transcripts of membrane progestin receptor α in the UVJ, and the translated proteins were detected in the UVJ extracts by Western blotting. Moreover, the number of secretory granules in the SST epithelial cells fluctuates during the ovulatory cycle, and the progesterone administration mimics this phenomena. A binding assay using [(3)H]-progesterone indicated the presence of a high affinity, limited capacity, saturable and single binding site for [(3)H]-progesterone in the membrane fraction of the UVJ, and this receptor did not interact with the synthetic antiprogestin RU486. These results demonstrated for the first time that the progesterone stimulates the release of the resident sperm from the SST and that the release of the sperm might occur via membrane progestin receptor α-mediating signal transduction.
Endocrinology 08/2011; 152(10):3952-62. · 4.46 Impact Factor
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ABSTRACT: The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. The glycoprotein meshwork of the IPVL forms a 3-dimensional matrix and possesses important functions in the fertilization process: it contributes to the binding of avian spermatozoa to the oocyte and induces acrosomal exocytosis. In contrast to the zona pellucida of mammals, the IPVL does not prevent the physiological polyspermy found in birds. Previous studies have shown that in the Japanese quail (Coturnix japonica) at least 5 glycoproteins are constituents of the IPVL (ZP1, ZP2, ZP3, ZP4, and ZPD). In this study, we investigated the spatiotemporal assembly pattern of the IPVL during folliculogenesis using immunohistochemical and ultrastructural methods. The obtained results clearly show that these glycoproteins are incorporated into the IPVL at distinct points during follicular development, supporting the hypothesis that ZP2 and ZP4 form a type of prematrix into which ZP1, ZP3, and ZPD are integrated at a later stage of development.
Cells Tissues Organs 07/2011; 195(4):330-9. · 2.20 Impact Factor
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ABSTRACT: An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45 kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45 kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.
Reproduction 06/2011; 142(2):267-76. · 2.58 Impact Factor
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ABSTRACT: The avian perivitelline layer, an extracellular matrix homologous to the zona pellucida (ZP) of mammalian oocytes, is composed mainly by zona pellucida gene family glycoproteins. Our previous studies in Japanese quail have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver. Recently, we demonstrated that another minor constituent, ZP2 is produced in the oocytes of the immature follicles. In the present study, we report the isolation of complementary DNA encoding quail ZP4 and its expression and origin in the female birds. By ribonuclease protection assay and in situ hybridization, we demonstrated that ZP4 transcripts were transcribed in the oocytes of small white follicles. The expression level of ZP4 decreased dramatically during follicular development, and the highest expression was observed in the small white follicles. Western blot analysis using the specific antibody against ZP4 indicated that the immunoreactive 58.2 kDa protein was present in the lysates of the small white follicles. These results demonstrate for the first time that the avian ZP4 is expressed in the oocyte, and that the expression pattern of the gene is similar to that of ZP2.
Animal Science Journal 04/2011; 82(2):227-35. · 0.86 Impact Factor
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ABSTRACT: This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.
Biology of Reproduction 12/2010; 83(6):965-9. · 4.01 Impact Factor
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ABSTRACT: In avian species, it has been assumed that an Fc receptor in the ovarian follicles mediates immunoglobulin Y (IgY) transport into the yolk. However, no such receptor responsible for IgY has been identified to date. To examine potential IgY binding activity in the entire ovarian follicle, whole-mount sections of quail ovarian follicle were incubated with the Fc fragment of chicken IgY (cIgY). Whole-mount frozen sections of the second largest ovarian follicle were prepared, and then the sections were incubated with digoxigenin-labeled Fc or Fab fragments of cIgY. Microscopic observation revealed that incubation with the cIgY-Fc fragment produced a binding signal in the inner layer of the ovarian follicular tissues, most likely in the granulosa cell layer. However, no such signal was detected when the sections were incubated with cIgY-Fab. Coincubation of the ovarian sections with Alexa488-labeled cIgY-Fc and antiserum raised against ZP1, an envelope protein specifically localized in the perivitelline layer, demonstrated that the source of the Fc binding signals partly coincided with the perivitelline layer. In conclusion, our data suggest that potential IgY binding substances interacting with the Fc domain are present in the inner layers of ovarian follicular tissues, most likely in the granulosa cell layer and/or in the perivitelline layer.
Animal Science Journal 10/2010; 81(5):580-5. · 0.86 Impact Factor
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Mihoko Kinoshita,
Daniela Rodler,
Kenichi Sugiura,
Kayoko Matsushima,
Norio Kansaku,
Kenichi Tahara,
Akira Tsukada,
Hiroko Ono,
Takashi Yoshimura,
Norio Yoshizaki,
Ryota Tanaka,
Tetsuya Kohsaka, Tomohiro Sasanami
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ABSTRACT: The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.
Reproduction 10/2009; 139(2):359-71. · 2.58 Impact Factor
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ABSTRACT: Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.
General and Comparative Endocrinology 05/2009; 161(2):238-45. · 3.27 Impact Factor
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ABSTRACT: In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.
FEBS Journal 08/2008; 275(14):3580-9. · 3.79 Impact Factor
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ABSTRACT: The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. The PL is constructed with two major glycoproteins, ZPC and ZP1, which are synthesized in the ovarian granulosa cells and the liver, respectively. Although the properties of the major components in the PL have been examined, knowledge about the nature of its minor constituents is lacking. In this study we focused on PL protein, which migrates at 46-kDa in the gel of SDS-PAGE. N-terminal sequence analysis demonstrated that the 46-kDa protein is the C-terminal fragment of ZP1. Analysis of lysylendopeptidase digests or cyanogens bromide-degraded fragments of ZP1 confirmed this postulate. Western blot analysis using antiserum against 46-kDa protein indicated the absence of 46-kDa protein in the serum. Moreover, small immunoreactive bands, thought to be cleaved fragments of ZP1, were detected in the PL lysate by western blot analysis using antiserum against the N-terminal peptide of ZP1. These results indicated that the N-terminal proteolytic processing of ZP1 might take place after the arrival of ZP1 at the ovary, and the resulting product, 46-kDa protein, is incorporated into the PL.
Animal Science Journal 01/2008; 79(1):104 - 110. · 0.86 Impact Factor
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ABSTRACT: Avian perivitelline membrane protein, ZP1, is synthesized and secreted by the liver with the stimulation of estrogens. In the present study, we measured the expression of ZP1 gene in the liver of immature male quail treated with various estrogenic compounds and in the liver of male quail embryos that were developed in the fertilized eggs laid by mother quail injected with various estrogenic compounds during vitellogenesis. Total RNA extracted from the liver was reverse-transcribed and cDNA was subjected to real-time PCR. Both diethylstilbestrol and ethinyl estradiol caused significant effect on the increase in mRNA in immature male quail. In contrast, diethylstilbestrol administered via the route of maternal injection was not effective for induction of embryonic mRNA, although the effect of ethinyl estradiol administered via the same route was prominent. These results showed that direct administration of estrogenic compounds, diethylstilbestrol and ethinyl estradiol, stimulates the induction of ZP1 gene, but the rate of accumulation of these compounds in the yolk is different during vitellogenesis. The present studies suggest that although ZP1 gene is a sensitive biomarker to evaluate the effects of endocrine disruptors, the route of administration is an important factor to compare the effectiveness.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 02/2007; 144(4):356-62. · 2.62 Impact Factor
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ABSTRACT: The present paper aimed to characterize the substance forming the sperm-associated body (SB), to find its producing sites, and to show its functions in the fertilization of chicken. The SB was found both in between the inner and outer layers of vitelline membranes around eggs and in the oviductal infundibulum. Material from which the SB is constructed (SB substance) was isolated from the vitelline membranes. It was a hydrophobic protein with a molecular size of 570 kDa. X-ray microanalysis detected calcium in the aggregates of the SB substance. Immunofluorescence and immunoelectron microscopy showed that the substance was produced in secretory cells in the luminal epithelium of the oviductal infundibulum and was provided to the egg on and in its vitelline membrane. During incubation, the SB substance bound with spermatozoa in the posterior portion of their flagella. Holes and disks were found in the vitelline membranes of fertile eggs at a ratio of 1: 19-24. Over 94% of the holes were accompanied by SB. The presence of SB is necessary for fertile spermatozoa to make holes in the membrane and to enter the fertile egg.
Embryologia 02/2007; 49(1):39-48. · 2.21 Impact Factor
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ABSTRACT: The extracellular matrix surrounding avian oocytes, called the perivitelline membrane (PL), consists of at least two major glycoproteins, ZP3 and ZP1. Our previous study using Japanese quail had demonstrated that the PL obtained from the preovulatory follicles was incubated in vitro with spermatozoa, and perforations were observed. This result indicated that the PL might contain a constituent that possesses activity to initiate the acrosome reaction (AR) in quail. In order to elaborate upon our previous findings, we evaluated the effects of ZP3 and ZP1 on the induction of sperm AR in Japanese quail. Ejaculated sperm were incubated with or without the purified PL glycoprotein, and their acrosome status was determined based on the presence or absence of the acrosome. Treatment of spermatozoa with increasing doses of the purified monomeric ZP1 led to a concentration-dependent stimulation of AR. The purified dimeric ZP1 had similar effect. Moreover, we found that the ZP1-induced AR was significantly blocked by the digestion of the PL protein with PNGaseF. In contrast, the addition of purified ZP3 failed to induce AR at any doses tested. These results indicate that N-linked glycans on ZP1 play an important role in triggering the AR in Japanese quail.
Reproduction (Cambridge, England) 02/2007; 133(1):41-9. · 3.09 Impact Factor
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ABSTRACT: Very low-density apolipoprotein II (apoVLDL) is one of the constituents of yolk in avian eggs. The expression of the apoVLDL gene is highly specific to the liver in mature female birds during the egg-laying period but is stimulated by exogenous estrogens in immature male birds. In the present study, we compared the effects of two estrogenic compounds, diethylstilbestrol and ethinylestradiol, on the expression of apoVLDL mRNA in the liver of Japanese quail (Coturnix japonica). Three-week-old, immature male quail were treated with a single intraperitoneal injection of the estrogenic compounds, and the level of apoVLDL mRNA in the liver was measured by gene-specific real-time polymerase chain reaction. Diethylstilbestrol and ethinylestradiol had a similar effect on the level of mRNA, increasing it in a dose-dependent manner. Next, the levels of apoVLDL mRNA in the liver of male embryos, which were developed in fertile eggs laid by quail injected with the estrogenic compounds during yolk formation, were measured. Maternal exposure to ethinylestradiol caused an increase in the mRNA in embryos, whereas exposure to diethylstilbestrol had no effect. These results point out the importance of the route of administration to the evaluation of the estrogenic effects of endocrine disruptors in oviparous species.
Environmental Toxicology and Chemistry 06/2006; 25(5):1354-9. · 2.81 Impact Factor
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ABSTRACT: The present study describes the holes in the inner vitelline membrane of fertile eggs of the quail Coturnix japonica, which remain after the spermatozoa pass through. It was shown that the light-microscopically observable 'holes' correspond mostly to electron-microscopically defined 'disks', and, to a lesser extent (about 5%), real holes. Immunofluorescent staining of the vitelline membranes with an antiquail ZPC antiserum was used to discriminate the holes from the disks light-microscopically. Over 96% of holes were accompanied by calcium-coated sperm-associated bodies, indicating a close relationship between the two. There was no preferential localization of the disks, holes or sperm-associated bodies in the vitelline membrane around the egg. The sperm-associated bodies bound with the spermatozoa at the posterior end of sperm flagella. Incubation of the inner vitelline membranes, isolated from the largest follicles, with spermatozoa resulted in production only of the disks, whereas the holes (about 9%) were produced when the sperm-associated bodies were added to the system. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding to the inner vitelline membrane, making holes in the membrane and passing through them in fertile eggs.
Embryologia 02/2006; 48(1):33-40. · 2.21 Impact Factor
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ABSTRACT: The extracellular matrix surrounding avian oocytes, referred to as the perivitelline membrane (PL), exhibits a three-dimensional network of fibrils between granulosa cells and the oocyte. We previously reported that one of its components, ZPC, is synthesized in granulosa cells that are specifically incorporated into the PL; this incorporation might be mediated by a specific interaction with ZPB1, another PL constituent, which is synthesized in the liver. In order to extend our previous findings, we established an expression system for quail ZPB1 using a mammalian cell line, and several ZPB1 mutants lacking the zona pellucida (ZP) domain or the glutamine-rich repeat region were produced. Western blot analysis of the immunoprecipitated materials with anti-ZPC antiserum indicated that ZPB1 was coimmunoprecipitated with the antiserum in the presence of ZPC. Ligand blotting also revealed the specific binding of ZPC and ZPB1 and indicated that the binding of these two components might be mediated via an ionic interaction. An analysis using recombinant ZPB1 demonstrated that the ZPB1 lacking the ZP domain did not bind to ZPC, whereas the mutant missing the glutamine-rich repeat region retained its capacity for binding. Furthermore, although the ZPB1 lacking the N-terminal half of the ZP domain was able to bind to ZPC, the deletion of the C-terminal half completely abolished ZPB1 binding to ZPC. These results suggested that the C-terminal half of the ZP domain of ZPB1 contains a binding site for ZPC, and that it appears to be involved in insoluble PL fibril formation in the quail ovary.
Cells Tissues Organs 02/2006; 183(1):41-52. · 2.20 Impact Factor
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ABSTRACT: The avian perivitelline membrane (PL), which is an investment homologous to the mammalian zona pellucida, is found between the surface of the oocyte and the apical surface of ovarian granulosa cells. Our previous study demonstrated that ZPC, one of the components of PL, is synthesized in ovarian granulosa cells. However, how the secretion of ZPC is regulated in the cells has been insufficiently investigated. We studied the secretion of quail ZPC expressed in polarized Madin-Darby canine kidney (MDCK) cells in a dual-chamber apparatus. Western blot analyses of the conditioned medium demonstrated that the majority of the secreted ZPC were distributed in the apical compartment. When ZPC lacking N-linked oligosaccharides was transfected into the cells, the 31-kDa immunoreactive band was detected in both the apical and the basolateral medium. Interestingly, immunohistochemical observations of the follicular wall demonstrated that the predominant intracellular form of ZPC in the cells localized in the apical side of the perinuclear region apposed to the PL, but not the basolateral side, indicating the possibility that ZPC could be selectively transported toward the apical surface in vivo. Taken together, these results indicated that ZPC expressed in MDCK cells are selectively released to the apical compartment, and that the N-linked carbohydrates might possess information that causes the efficient transport of ZPC to the apical surface of the cells.
Cells Tissues Organs 02/2005; 180(3):169-77. · 2.20 Impact Factor
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ABSTRACT: The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. We have previously reported that one of its components, ZPC, is produced in ovarian granulosa cells by the stimulation of follicle-stimulating hormone and testosterone. Another component, ZP1, is synthesized in the liver and might be transported to the surface of the oocyte of the follicles. These glycoproteins are assembled to form a three-dimensional network of coarse fibers between the granulosa cells and the oocyte. In the present study, we have evaluated the involvement of the interaction of ZPC and ZP1 in the formation of the PL of Japanese quail. By measuring the incorporation of tritium-labeled proteins into the PL, we have found that tritium-labeled ZPC is specifically incorporated into the PL. Whole-mount autoradiographic analysis of the PL has also revealed the incorporation of the secreted ZPC into the isolated PL. To study which component in the PL is responsible for the specific incorporation of ZPC, PL lysates were incubated with the conditioned medium of the granulosa cells and were immunoprecipitated with anti-ZPC antiserum. Western blot analysis of the immunoprecipitated materials indicated that the 175-kDa and 97-kDa ZP1 forms were co-immunoprecipitated with anti-ZPC antiserum. These results demonstrate that ZPC secreted from the granulosa cells specifically binds with ZP1, and that the phenomenon might be involved in insoluble PL fiber formation in quail ovary.
Cell and Tissue Research 01/2005; 318(3):565-70. · 3.11 Impact Factor
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Journal of Poultry Science - J POULT SCI. 01/2004; 41(1):30-37.
