Cortical granule biogenesis is active throughout oogenesis in sea urchins

Development ◽  
1994 ◽  
Vol 120 (5) ◽  
pp. 1325-1333 ◽  
Author(s):  
M. Laidlaw ◽  
G.M. Wessel
Keyword(s):  
Recombinant Proteins ◽  
Sea Urchins ◽  
Germinal Vesicle ◽  
Cortical Granule ◽  
Cdna Clones ◽  
Secretory Vesicles ◽  
Cortical Granules ◽  
Oocyte Growth ◽  
Peak Abundance ◽  
Cognate Protein

Cortical granules are secretory vesicles formed in the eggs of most animals and are essential for the prevention of polyspermy in these organisms. We have studied the biogenesis of cortical granules in sea urchin oocytes by identifying cDNA clones that encode proteins targeted selectively to the cortical granules. These cDNA clones were identified by an immunoscreen of a cDNA library using antibodies to proteins of the fertilization envelope. Four different mRNAs were identified, ranging from 4 kb to 13 kb in length, that encoded proteins targeted specifically to cortical granules. Accumulation of these mRNAs began very early in oogenesis, in oocytes approximately 10–15 microns in diameter, and continued throughout oogenesis. The mRNAs reached peak abundance (on a per cell basis) in germinal vesicle stage oocytes, and the accumulation of each mRNA was linear with respect to oocyte growth. During breakdown of the germinal vesicle these mRNAs were degraded so that in eggs the mRNA signals were at background levels. Antibodies generated to recombinant proteins made from each of these cDNA clones showed that in the oocyte each cognate protein appeared early in oogenesis. These proteins accumulated only in cortical granules: no accumulation was seen in the cytoplasm, in Golgi, or in other vesicles, and no heterogeneity of the contents was seen within the population of cortical granules. Using these antibodies we show that cortical granules accumulated linearly throughout oogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Regulated Proteolysis by Cortical Granule Serine Protease 1 at Fertilization

2004 ◽  
Vol 15 (5) ◽  
pp. 2084-2092 ◽  
Author(s):  
Sheila A. Haley ◽  
Gary M. Wessel
Keyword(s):  
Serine Protease ◽  
Protease Activity ◽  
Sea Urchins ◽  
Small Population ◽  
Structural Proteins ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Fluorescent Indicators ◽  
Seawater Ph ◽  
Protease Activation

Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are β-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.

scholarly journals Rendezvin: An Essential Gene Encoding Independent, Differentially Secreted Egg Proteins That Organize the Fertilization Envelope Proteome after Self-Association

2006 ◽  
Vol 17 (12) ◽  
pp. 5241-5252 ◽  
Author(s):  
Julian L. Wong ◽  
Gary M. Wessel
Keyword(s):  
Extracellular Matrix ◽  
Sea Urchins ◽  
Essential Gene ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Gene Encoding ◽  
Self Association ◽  
Granule Secretion

Preventing polyspermy during animal fertilization relies on modifications to the egg's extracellular matrix. On fertilization in sea urchins, the contents of cortical granules are secreted and rapidly assemble into the egg's extracellular vitelline layer, forming the fertilization envelope, a proteinaceous structure that protects the zygote from subsequent sperm. Here, we document rendezvin, a gene whose transcript is differentially spliced to yield proteins destined for either cortical granules or the vitelline layer. These distinctly trafficked variants reunite after cortical granule secretion at fertilization. Together, they help coordinate assembly of the functional fertilization envelope, whose proteome is now defined in full.

scholarly journals Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems

2015 ◽  
Vol 27 (7) ◽  
pp. 1082 ◽  
Author(s):  
Maricy Apparicio ◽  
Giuliano Q. Mostachio ◽  
Tathiana F. Motheo ◽  
Aracelle E. Alves ◽  
Luciana Padilha ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
System A ◽  
Cytoplasmic Maturation ◽  
Positive Effect

The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72 h in base medium (BM) with 10 IU mL–1 human chorionic gonadotrophin (hCG), 1 μg mL–1 progesterone (P4) and 1 μg mL–1 oestradiol (E2); in bi-phasic System B oocytes were matured for 48 h in BM with hCG and for 24 h in BM with P4; in bi-phasic System C oocytes were matured for 48 h in BM with hCG, P4 and E2, and for 24 h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (P < 0.05). Bi-phasic systems were also beneficial for cortical granule distribution (an indication of cytoplasmic maturation) and its relationship to nuclear status: 74.5% of the oocytes cultured in System B and 85.4% of those cultured in System C presented both nuclear and cytoplasmic maturation (P < 0.001). The stage of the oestrous cycle did not influence maturation rates.

scholarly journals Knockin’ on Egg’s Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species

2021 ◽  
Vol 9 ◽  
Author(s):  
Japhet Rojas ◽  
Fernando Hinostroza ◽  
Sebastián Vergara ◽  
Ingrid Pinto-Borguero ◽  
Felipe Aguilera ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Animal Species ◽  
Egg Activation ◽  
Cortical Granule ◽  
Secretory Vesicles ◽  
Cortical Granules ◽  
Maternal Control ◽  
Molecular Machinery ◽  
Cortical Granule Exocytosis ◽  
Critical Aspects

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

236 NUCLEAR AND CYTOPLASMIC MODIFICATIONS OF GERMINAL VESICLE BOVINE OOCYTES IN RELATION TO CHROMATIN REMODELING

2006 ◽  
Vol 18 (2) ◽  
pp. 226
Author(s):  
V. Lodde ◽  
P. Maddox-Hyttel ◽  
S. Modina ◽  
A. M. Luciano
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cortical Granules ◽  
Oocyte Growth ◽  
Structural Modifications ◽  
Chromatin Configuration ◽  
Hoechst Staining ◽  
Bovine Oocytes

We previously reported that germinal vesicle (GV) bovine oocytes can be classified on the basis of their chromatin organization and that increased chromatin condensation is accompanied by a major incidence of gap junction-mediated coupling interruption between germ and cumulus cells and by an increase in oocyte developmental competence (Lodde et al. 2005 Reprod. Fertil. Dev. 17(2), 294-295). The aim of this study was to characterize, at the ultrastructural level, both nuclear and cytoplasmic compartments of bovine oocytes classified according to their chromatin configuration because key structural modifications, such as nucleolar inactivation and remodeling of specific ooplasmic structures, take place during the later phases of oocyte growth. Cumulus-oocyte complexes collected from 0.5-2-mm early antral (EA) and 2-6-mm mid-antral (MA) follicles were freed of cumulus cells. Denuded oocytes were stained with Hoechst 33342, classified according to the degree of chromatin condensation, and processed for light microscopy of semi-thin sections (LM; n = 10 in each class) and transmission electron microscopy (TEM; n = 5 in each class). Four classes of oocytes were identified by the Hoechst staining: GV0 with filamentous chromatin diffused in the nuclear area, GV1 with few foci of condensed chromatin, GV2 with chromatin further condensed into distinct clumps, and GV3 with chromatin condensed into a single clump. Almost all oocytes collected from EA follicles were classified as GV0. Oocytes of this class were absent in MA follicles, whereas class GV1, GV2, and GV3 oocytes occurred at similar frequency. LM confirmed the chromatin condensation found by the Hoechst staining and revealed that in class GV2 and GV3 oocytes the chromatin was mainly located close to the nucleolus. Ultrastructurally, the nucleolus was fibrillo-granular in GV0 oocytes; the oocytes in the other classes displayed an electron dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most organelles were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex, were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Our findings indicate that the process of chromatin remodeling is strictly related to structural modifications that characterize the later stages of the oocyte growth phase. Because the highest degree of chromatin condensation was combined with degenerative features of cortical granules, we hypothesize that this class of oocytes (GV3) originated from early atretic follicles, as also suggested in other species. The evaluation of oocytes on the basis of chromatin configuration may be useful for the development of new strategies for manipulating fertility in mammals. This work was supported by a COFIN Grant.

Microfilament assembly and cortical granule distribution during maturation, parthenogenetic activation and fertilisation in the porcine oocyte

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 145-149 ◽  
Author(s):  
Nam-Hyung Kim ◽  
Billy N. Day ◽  
Hoon Taek Lee ◽  
Kil-Saeng Chung
Keyword(s):  
Laser Scanning ◽  
Germinal Vesicle ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Cortex ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
Parthenogenetic Activation ◽  
Scanning Confocal Microscopy

SummaryIn this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.

Cortical granules of the sea urchin translocate early in oocyte maturation

Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1845-1850
Author(s):  
L.K. Berg ◽  
G.M. Wessel
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown

Cortical granules are secretory vesicles poised at the cortex of an egg that, upon stimulation by sperm contact at fertilization, secrete their contents. These contents modify the extracellular environment and block additional sperm from reaching the egg. The role of cortical granules in blocking polyspermy is conserved throughout much of phylogeny. In the sea urchin, cortical granules accumulate throughout the cytoplasm during oogenesis, but in mature eggs the cortical granules are attached to the plasma membrane, having translocated to the cortex at some earlier time. To study the process of cortical granule translocation to the cell surface we have devised a procedure for maturation of sea urchin oocytes in vitro. Using this procedure, we examined the rate of oocyte maturation by observing the movement and breakdown of the germinal vesicle, the formation of polar bodies and the formation of the egg pronucleus. We find that oocyte maturation takes approximately 9 hours in the species used here (Lytechinus variegatus), from the earliest indication of maturation (germinal vesicle movement) to formation of a distinct pronucleus. We then observed the translocation of cortical granules in these cells by immunolocalization using a monoclonal antibody to hyalin, a protein packaged specifically in cortical granules. We found that the translocation of cortical granules in in vitro-matured oocytes begins with the movement of the germinal vesicle to the oocyte cell surface, and is 50% complete 1 hour after germinal vesicle breakdown. In the in vitro-matured egg, 99% of the cortical granules are at the cortex, indistinguishable from translocation in oocytes that mature in vivo. We have also found that eggs that mature in vitro are functionally identical to eggs that mature in vivo by four criteria. (1) The matured cells undergo a selective turnover of mRNA encoding cortical granule contents. (2) The newly formed pronucleus begins transcription of histone messages. (3) Cortical granules that translocate in vitro are capable of exocytosis upon activation by the calcium ionophore, A23187. (4) The mature egg is fertilizable and undergoes normal cleavage and development. In vitro oocyte maturation enables us to examine the mechanism of cortical granule translocation and other processes that had previously only been observed in static sections of fixed ovaries.

Cortical changes in starfish (Asterina pectinifera) oocytes during 1-methyladenine-induced maturation and fertilisation/activation

Zygote ◽  
1995 ◽  
Vol 3 (3) ◽  
pp. 225-239 ◽  
Author(s):  
Frank J. Longo ◽  
Mark Woerner ◽  
Kazuyoshi Chiba ◽  
Motonori Hoshi
Keyword(s):  
Germinal Vesicle ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes ◽  
A 23187 ◽  
Asterina Pectinifera ◽  
Cortical Maturation ◽  
Three Phases

SummaryMaturation of the starfish oocyte cortex to produce an effective cortical granule reaction and fertilisation envelope is believed to develop in three phases: (1) pre-methyladenine (1-MA) stimulation; (2) post-1-MA stimulation, pregerminal vesicle breakdown; and (3) post-germinal vesicle breakdown. The present study was initiated to identify what each of these phases may encompass, specifically with respect to structures associated with the oocyte cortex, including cortical granules, microvilli and vitelline layer. 1-MA treatment brought about an orientation of cortical granules such that they became positioned perpendicular to the oocyte surface, and an ∼ 4-fold decrease in microvillar length. A-23187 activation of immature oocytes treated with (10 min; pregerminal vesicle breakdown) or without 1-MA resulted in a reduction in cortical granule number of 21% and 41%, respectively (mature oocytes underwent a 96% reduction in cortical granules). Elevation of the fertilisation envelope in both cases was significantly retarded compared with activated mature oocytes. In activated mature oocytes, the vitelline layer elevated 20.0 ± 5.4 μm from the egg's surface, whereas in immature oocytes treated with just A-23187 or with 1-MA (10 min) and A-23187, it lifted 0.35 ± 0.1 and 0.17 ± 0.04 μm, respectively. The fertilisation envelopes of activated (or fertilised) immature oocytes also differed morphologically from those of mature oocytes. In activated, immature oocytes, the fertilisation envelope was not uniform in its thickness and possessed thick and thin regions as well as fenestrations. Additionally, it lacked a complete electron-dense stratum that characterised the fertilisation envelopes of mature oocytes. The nascent perivitelline space of immature oocytes was also distinguished by the presence of numerous vesicles which appeared to be derived from microvilli. Differences in the morphology of cortices from activated (fertilised) and non-activated, immature and mature oocytes substantiate previous investigations demonstrating three phases of cortical maturation, and are consistent with physiological changes that occur during oocyte maturation, involving ionic conductance of the plasma membrane, establishment of slow and fast blocks to polyspermy and elevation of a fertilisation envelope.

Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte

Development ◽  
2002 ◽  
Vol 129 (18) ◽  
pp. 4315-4325
Author(s):  
Gary M. Wessel ◽  
Sean D. Conner ◽  
Linnea Berg
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Time Window ◽  
Germinal Vesicle ◽  
Mature Oocyte ◽  
Meiotic Maturation ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
Translocation Event

Cortical granules exocytose after the fusion of egg and sperm in most animals, and their contents function in the block to polyspermy by creating an impenetrable extracellular matrix. Cortical granules are synthesized throughout oogenesis and translocate en masse to the cell surface during meiosis where they remain until fertilization. As the mature oocyte is approximately 125 μm in diameter (Lytechinus variegatus), many of the cortical granules translocate upwards of 60 μm to reach the cortex within a 4 hour time window. We have investigated the mechanism of this coordinated vesicular translocation event. Although the stimulus to reinitiate meiosis in sea urchin oocytes is not known, we found many different ways to reversibly inhibit germinal vesicle breakdown, and used these findings to discover that meiotic maturation and cortical granule translocation are inseparable. We also learned that cortical granule translocation requires association with microfilaments but not microtubules. It is clear from endocytosis assays that microfilament motors are functional prior to meiosis, even though cortical granules do not use them. However, just after GVBD, cortical granules attach to microfilaments and translocate to the cell surface. This latter conclusion is based on organelle stratification within the oocyte followed by positional quantitation of the cortical granules. We conclude from these studies that maturation promoting factor (MPF) activation stimulates vesicle association with microfilaments, and is a key regulatory step in the coordinated translocation of cortical granules to the egg cortex.

Oocyte maturation: aberrant post-fusion responses of the rabbit primary oocyte to penetrating spermatozoa

1979 ◽  
Vol 39 (1) ◽  
pp. 1-12
Author(s):  
M. Berrios ◽  
J.M. Bedford
Keyword(s):  
Anterior Part ◽  
Light And Electron Microscopy ◽  
Germinal Vesicle ◽  
Sperm Chromatin ◽  
Fallopian Tubes ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
Acrosomal Membrane ◽  
Primary Oocyte ◽  
Inner Acrosomal Membrane

Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated previously with spermatozoa, recovered 2–5 h later and examined by light and electron microscopy. At least 4 aspects of GV oocyte/sperm interaction were abnormal. Although the vestments and oolemma seem normally receptive to spermatozoa, fusion with the oolemma of the primary oocyte did not elicit exocytosis of cortical granules, and consequently multiple entry of spermatozoa into the ooplasm was common. Secondly, the GV oocyte cortex failed to achieve a normal englufment of the anterior part of the sperm head. It sank into the ooplasm capped by only a small rostral vesicle or left the stable inner acrosomal membrane as a patch in the oolemma. Only rarely then was there significant dispersion of the sperm chromatin, and this remained surrounded by nuclear envelope. The persistence of this envelope constitutes a further aberrant feature, for it disappears immediately in secondary oocytes and was absent in primary oocytes in which germinal vesicle breakdown had occurred. The results are discussed with particular reference to current ideas about male pronucleus formation.

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