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Cite this page: Hill, M.A. (2019, October 19) Embryology Zona pellucida. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Talk:Zona_pellucida
Fertil Steril. 2019 Mar 25. pii: S0015-0282(19)30081-0. doi: 10.1016/j.fertnstert.2019.02.003. [Epub ahead of print] Characterization of follicles in girls and young women with Turner syndrome who underwent ovarian tissue cryopreservation. Mamsen LS1, Charkiewicz K2, Anderson RA3, Telfer EE4, McLaughlin M4, Kelsey TW5, Kristensen SG6, Gook DA7, Ernst E8, Andersen CY6. Author information Abstract OBJECTIVE: To characterize ovarian follicles of girls and young women with Turner syndrome (TS) who underwent ovarian tissue cryopreservation (OTC).
DESIGN: Retrospective case-control study.
SETTING: University hospital.
PATIENT(S): Fifteen girls and young women with TS aged 5-22 years at OTC were included, together with 42 control girls and young women aged 1-25 years who underwent OTC because of cancer.
MAIN OUTCOME MEASURE(S): Follicle density (follicles/mm3), morphology, and health were assessed in ovarian cortex biopsies from TS patients and compared with controls. Hormone concentrations were measured in serum and follicle fluids. Immature cumulus oocyte complexes were obtained and matured in vitro.
RESULT(S): Follicles were found in 60% of the biopsies (9 of 15) from TS ovaries. In 78% of the ovaries (7 of 9) with follicles, the follicle density was within the 95% confidence interval of the control group. There was a high rate of abnormal follicle morphology. Six follicle-specific proteins were expressed similarly in TS and control ovaries. However, apoptosis and zona pellucida protein expression were found to be abnormal in TS. Turner syndrome follicle fluid from small antral follicles had lower concentrations of estrogen and testosterone and higher concentrations of antimüllerian hormone than controls. Thirty-one cumulus oocyte complexes were collected from one patient and cultured for 48 hours in vitro, resulting in five metaphase II oocytes (maturation rate 16%, degeneration rate 19%).
CONCLUSION(S): The benefits of OTC may be limited to a highly selected group of TS mosaic patients in whom a sizeable pool of normal follicles is present at OTC.
Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
KEYWORDS: Turner syndrome; follicle density; ovarian tissue cryopreservation PMID: 30922638 DOI: 10.1016/j.fertnstert.2019.02.003
Curr Top Dev Biol. 2018;130:413-442. doi: 10.1016/bs.ctdb.2018.02.007. Epub 2018 May 10. Structure of Zona Pellucida Module Proteins. Bokhove M1, Jovine L2. Author information Abstract The egg coat, an extracellular matrix made up of glycoprotein filaments, plays a key role in animal fertilization by acting as a gatekeeper for sperm. Egg coat components polymerize using a common zona pellucida (ZP) "domain" module that consists of two related immunoglobulin-like domains, called ZP-N and ZP-C. The ZP module has also been recognized in a large number of other secreted proteins with different biological functions, whose mutations are linked to severe human diseases. During the last decade, tremendous progress has been made toward understanding the atomic architecture of the ZP module and the structural basis of its polymerization. Moreover, sperm-binding regions at the N-terminus of mollusk and mammalian egg coat subunits were found to consist of domain repeats that also adopt a ZP-N fold. This discovery revealed an unexpected link between invertebrate and vertebrate fertilization and led to the first structure of an egg coat-sperm protein recognition complex. In this review we summarize these exciting findings, discuss their functional implications, and outline future challenges that must be addressed in order to develop a comprehensive view of this family of biomedically important extracellular molecules.
Copyright © 2018 Elsevier Inc. All rights reserved.
KEYWORDS: Endoglin–BMP9 complex; Fertilization; Uromodulin; VERL–lysin complex; X-ray crystallography; ZP domain; ZP module; ZP-C domain; ZP-N domain; ZP1; ZP2; ZP3; ZP4; Zona pellucida PMID: 29853186 DOI: 10.1016/bs.ctdb.2018.02.007
ZP3 is Required for Germinal Vesicle Breakdown in Mouse Oocyte Meiosis
Sci Rep. 2017 Feb 1;7:41272. doi: 10.1038/srep41272.
Gao LL1, Zhou CX1, Zhang XL1, Liu P1, Jin Z1, Zhu GY1, Ma Y1, Li J1, Yang ZX1, Zhang D1.
ZP3 is a principal component of the zona pellucida (ZP) of mammalian oocytes and is essential for normal fertility, and knockout of ZP3 causes complete infertility. ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction; however, additional cellular roles for ZP3 in mammalian oocytes have not been yet reported. In the current study, we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP. Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown (GVBD) (marking the beginning of meiosis), significantly reducing the percentage of MII oocytes. To investigate the ZP3-mediated mechanisms governing GVBD, we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry. We identified Protein tyrosine phosphatase, receptor type K (Ptprk), Aryl hydrocarbon receptor-interacting protein-like 1 (Aipl1), and Diaphanous related formin 2 (Diaph2) as potential candidates, and established a working model to explain how ZP3 affects GVBD. Finally, we provided preliminary evidence that ZP3 regulates Akt phosphorylation, lamin binding to the nuclear membrane via Aipl1, and organization of the actin cytoskeleton via Diaph2. These findings contribute to our understanding of a novel role played by ZP3 in GVBD. PMID: 28145526 PMCID: PMC5286536 DOI: 10.1038/srep41272
Perivitelline threads in cleavage-stage human embryos: observations using time-lapse imaging
Reprod Biomed Online. 2017 Sep 28. pii: S1472-6483(17)30452-2. doi: 10.1016/j.rbmo.2017.09.004. [Epub ahead of print]
Kellam L1, Pastorelli LM2, Bastida AM1, Senkbeil A1, Montgomery S3, Fishel S1, Campbell A4.
Time-lapse imaging of the human preimplantation embryo in vitro has revealed a transient phenomenon involving the appearance of perivitelline threads, commonly observed at the two-cell stage. These threads span the perivitelline space, arising at the specific area where the cytoplasmic membrane contacts the zona pellucida, before any perivitelline space is formed. The threads persist as the cytoplasmic membrane retracts from the zona pellucida to form the first cleavage furrow. In this observational report, these structures and their incidence are described. A total of 834 time-lapse videos from IVF treatment cycles, one per patient, were retrospectively analysed for perivitelline threads, from pronuclear formation until completion of the first cell cycle. Threads were observed in 56.4% (470/834) of embryos and varied from a single to an array spanning an area of the zona pellcida. A total of 91.9% (432/470) were seen to form after cytoplasmic membrane-zona-pellucida contact. A total of 76.4% (359/470) were visible at the first cleavage furrow; 77% (362/470) were associated with cytoplasmic fragments at the two-cell-stage. Presence or absence of threads did not affect embryo development. This descriptive study is limited; further characterization of these structures is needed to elucidate their potential role in early human embryo development. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: Cleavage; Cytokinesis; Fragmentation; Perivitelline space; Zona pellucida; Zygote
PMID: 29074360 DOI: 10.1016/j.rbmo.2017.09.004
A Bespoke Coat for Eggs: Getting Ready for Fertilization
Curr Top Dev Biol. 2016;117:539-52. doi: 10.1016/bs.ctdb.2015.10.018. Epub 2016 Jan 7.
Wassarman PM1, Litscher ES2.
All eggs have an extracellular coat (EC) that plays unique roles during reproduction and development. ECs are designed to protect eggs and support their growth, regulate fertilization of eggs, and protect early embryos. ECs of mammalian and nonmammalian eggs consist of only a few proteins that are closely related to one another. All these proteins possess regulatory elements and a structural domain responsible for processing and assembly of the proteins into ECs. They also possess regions responsible for their functional roles during and after fertilization. Our essay addresses these and other aspects of EC biology. © 2016 Elsevier Inc. All rights reserved. KEYWORDS: Eggs; Extracellular coat; Fertilization; Vitelline envelope; Zona pellucida; Zona pellucida domain; Zona pellucida proteins
A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans
J Cell Biol. 2014 Jun 23;205(6):801-9. doi: 10.1083/jcb.201404025. Epub 2014 Jun 16.
Avella MA1, Baibakov B1, Dean J2.
Abstract The extracellular zona pellucida surrounds ovulated eggs and mediates gamete recognition that is essential for mammalian fertilization. Zonae matrices contain three (mouse) or four (human) glycoproteins (ZP1-4), but which protein binds sperm remains controversial. A defining characteristic of an essential zona ligand is sterility after genetic ablation. We have established transgenic mice expressing human ZP4 that form zonae pellucidae in the absence of mouse or human ZP2. Neither mouse nor human sperm bound to these ovulated eggs, and these female mice were sterile after in vivo insemination or natural mating. The same phenotype was observed with truncated ZP2 that lacks a restricted domain within ZP2(51-149). Chimeric human/mouse ZP2 isoforms expressed in transgenic mice and recombinant peptide bead assays confirmed that this region accounts for the taxon specificity observed in human-mouse gamete recognition. These observations in transgenic mice document that the ZP2(51-149) sperm-binding domain is necessary for human and mouse gamete recognition and penetration through the zona pellucida.
Structural analysis of peptide-analogues of human Zona Pellucida ZP1 protein with amyloidogenic properties: insights into mammalian Zona Pellucida formation
PLoS One. 2013 Sep 12;8(9):e73258. doi: 10.1371/journal.pone.0073258. eCollection 2013.
Louros NN1, Iconomidou VA, Giannelou P, Hamodrakas SJ.
Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as 'aggregation-prone' by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted 'aggregation-prone' parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization.
Investigation of the mechanisms by which the molecular chaperone HSPA2 regulates the expression of sperm surface receptors involved in human sperm-oocyte recognition
Mol Hum Reprod. 2012 Dec 17. [Epub ahead of print]
Redgrove KA, Anderson AL, McLaughlin EA, O'Bryan MK, Aitken RJ, Nixon B. Source Reproductive Science Group, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
A unique characteristic of mammalian spermatozoa is that upon ejaculation, they are unable to recognise and bind to an ovulated oocyte. These functional attributes are only realised following the cells' ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as 'capacitation'. We have previously shown that this functional transformation is, in part, engineered by the modification of the sperm surface architecture leading to the assembly and/or presentation of multimeric sperm-oocyte receptor complexes. In this study, we have extended our findings through the characterisation of one such complex containing arylsulfatase A (ARSA), sperm adhesion molecule 1 (SPAM1) and the molecular chaperone, heat shock protein 2A (HSPA2). Through the application of flow cytometry we revealed that this complex undergoes a capacitation-associated translocation to facilitate the repositioning of ARSA to the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida interactions. Conversely, SPAM1 appears to reorient away from the sperm surface, possibly reflecting its primary role in cumulus matrix dispersal preceding sperm-zona pellucida recognition. The dramatic relocation of the complex was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol or broad spectrum protein kinase A (PKA) and tyrosine kinase inhibitors suggesting that it may be driven by alterations in membrane fluidity characteristics and concurrently by the activation of a capacitation-associated signal transduction pathway. Collectively these data afford novel insights into the sub-cellular localization and potential functions of multimeric protein complexes in human spermatozoa.
Biosynthesis of hamster zona pellucida is restricted to the oocyte
Theriogenology. 2011 Feb;75(3):463-72. Epub 2010 Nov 12.
Izquierdo-Rico MJ, Gimeno L, Jiménez-Cervantes C, Ballesta J, Avilés M. Department of Cell Biology and Histology, Faculty of Medicine, University of Murcia, Murcia, Spain.
The zona pellucida (ZP) is an extracellular coat that surrounds the mammalian oocyte and the early embryo until implantation. This coat mediates several critical aspects of fertilization, including species-selective sperm recognition, the blocking of polyspermy and protection of the oocyte and the preimplantation embryo. Depending on the species, the ZP is composed of three to four different glycoproteins encoded by three or four genes. These genes have been cloned and sequenced for different species. However, controversy exists about the cell type specificity of the ZP glycoproteins, for which several models have been proposed. Different groups have reported that ZP is produced only by the oocytes, by the granulosa cells or by both cell types, depending on the species under study. We recently described the expression of four ZP proteins in the hamster ovary. By means of the complete set of the hamster ZP cDNAs, we undertook the study of the origin and expression pattern of the four ZP genes. In the present work, the expression of ZP1, ZP2, ZP3 and ZP4 is carefully analyzed by in situ hybridization (ISH) in hamster ovaries. Our data suggest that the four hamster ZP genes are expressed in a coordinate and oocyte-specific manner during folliculogenesis. Furthermore, this expression is maximal during the first stages of the oocyte development and declines in oocytes from later development stages, particularly within large antral follicles.
Copyright © 2011 Elsevier Inc. All rights reserved.
High doses of medroxyprogesterone as the cause of disappearance of adherence of the zona pellucida to an oocyte
Protoplasma. 2010 Oct;246(1-4):101-7. Epub 2010 Aug 17.
Jodłowska-Jedrych B, Jedrych M, Matysiak W. Source Department of Histology and Embryology, Medical University of Lublin, Radziwiłłowska 11, 20-080, Lublin, Poland. firstname.lastname@example.org
The zona pellucida (ZP) is an external glycoprotein membrane of oocytes of mammals and embryos in the early stage of their development. ZP first appears in growing ovarian follicles as an extracellular substance between the oocyte and granular cells. The zona pellucid markedly affects the development and maturation of the oocyte. The morphology of the ZP-oocyte complex allows a more precise determination of the oocyte maturity. According to numerous experimental studies, ZP is essential for preimplantation embryonic development of humans and other mammals. It prevents dispersion of blastomeres and enhances their mutual interactions. ZP is a dynamic structure responsible for the provision of nutrients to early forms of oocytes in mammals. The aim of the present study was untrastructural evaluation of the ZP-oocyte contact during inhibited ovulation. Female white rats (Wistar strain) received a suspension of medroxyprogesterone acetate (MPA) in incremental intramuscular bolus doses of 3.7 mg (therapeutic dose), 7.4 mg and 11.1 mg. The animals were decapitated 5 days after the administration of MPA. Ovarian sections were evaluated under a transmission electron microscope (TEM) Zeiss EM 900. Morphometric analysis of ZP was conducted using the cell imaging system by Olympus. In females exposed to therapeutic doses of MPA, ZP showed the structure of granular-fibrous reticulum of a medium electron density with single cytoplasmic processes originating from the surrounding structures. The oocyte cell membrane generated single, delicate processes directed toward ZP. Microvilli of the oocyte were short and thin. In the group receiving 7.4 mg of MPA, ZP had the structure of a delicate, loose granular-fibrous reticulum, and the oocyte cell membrane generated single microvilli directed toward ZP. In both those groups, the close ZP-oocyte contact was observed. Otherwise, in the group exposed to the highest MPA doses (11.1 mg), thicker and more numerous oocyte microvilli were found, which did not penetrate ZP matrix. They were dense, irregularly separated contour, forming a barrier between ZP and oocyte. The present findings are likely to suggest that MPA has inhibiting effects on the synthesis of binding proteins and causes the loss of the oocyte contact with ZP.
New insights into the mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs
Biol Reprod. 2009 Nov;81(5):856-70. Epub 2009 Jul 8.
Mugnier S, Dell'Aquila ME, Pelaez J, Douet C, Ambruosi B, De Santis T, Lacalandra GM, Lebos C, Sizaret PY, Delaleu B, Monget P, Mermillod P, Magistrini M, Meyers SA, Goudet G. Institut National de la Recherche Agronomique, UMR85, Physiologie de la Reproduction et des Comportements, Nouzilly, France.
The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.
PMID: 19587333 http://www.ncbi.nlm.nih.gov/pubmed/19587333
Zona pellucida glycoproteins
J Biol Chem. 2008 Sep 5;283(36):24285-9. Epub 2008 Jun 6.
Wassarman PM. Source Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. email@example.com Abstract All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida, that plays vital roles during oogenesis, fertilization, and preimplantation development. The mouse zona pellucida consists of three glycoproteins that are synthesized solely by growing oocytes and assemble into long fibrils that constitute a matrix. Zona pellucida glycoproteins are responsible for species-restricted binding of sperm to unfertilized eggs, inducing sperm to undergo acrosomal exocytosis, and preventing sperm from binding to fertilized eggs. Many features of mammalian and non-mammalian egg coat polypeptides have been conserved during several hundred million years of evolution.
Detection of zona pellucida proteins during human folliculogenesis
Hum Reprod. 2008 Feb;23(2):394-402. Epub 2007 Nov 22.
Gook DA, Edgar DH, Borg J, Martic M. Reproductive Services, Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. firstname.lastname@example.org
Abstract BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. METHODS: Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis. PMID: 18033806
All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.
Three-dimensional structure of the zona pellucida at ovulation
Microsc Res Tech. 2006 Jun;69(6):415-26.
Familiari G, Relucenti M, Heyn R, Micara G, Correr S. Laboratory of Electron Microscopy, Pietro M. Motta, Department of Anatomy, University of Rome La Sapienza, 00161 Rome, Italy. email@example.com
The mammalian zona pellucida (ZP) is an extracellular matrix surrounding oocytes and early embryos, which is critical for normal fertilization and preimplantation development. It is made up of three/four glycoproteins arranged in a delicate filamentous matrix. Scanning electron microscopy (SEM) studies have shown that ZP has a porous, net-like structure and/or nearly smooth and compact aspect. In this study, the fine 3-D structure of the human and mouse ZP is reviewed with the aim to integrate ultrastructural and molecular data, considering that the mouse is still used as a good model for human fertilization. By conventional SEM observations, numerous evidences support that the spongy ZP appearance well correlates with mature oocytes. When observed through more sophisticated techniques at high resolution SEM, ZP showed a delicate meshwork of thin interconnected filaments, in a regular alternating pattern of wide and tight meshes. In mature oocytes, the wide meshes correspond to "pores" of the "spongy" ZP, whereas the tight meshes correspond to the compact parts of the ZP surrounding the pores. In conclusion, the traditional "spongy" or "compact" appearance of the ZP at conventional SEM appears to be only the consequence of a prevalence of different arrangements of microfilament networks, according to the maturation stage of the oocyte, and in agreement with the modern supramolecular model of the ZP at the basis of egg-sperm recognition. Despite great differences in molecular characterization of ZP glycoproteins between human and mouse ZP, there are no differences in the 3-D organization of glycoproteic microfilaments in these species.
PMID: 16703610 http://www.ncbi.nlm.nih.gov/sites/entrez/16703610
Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes
Biol Reprod. 2002 Apr;66(4):866-76.
El-Mestrah M, Castle PE, Borossa G, Kan FW. Department of Anatomy and Cell Biology, Faculty of Health Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6.
The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group. PMID: 11906903
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