scholarly journals The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present

Reproduction ◽  
1990 ◽  
Vol 89 (1) ◽  
pp. 253-259 ◽  
Author(s):  
C. Vincent ◽  
S. J. Pickering ◽  
M. H. Johnson
Keyword(s):  
Zona Pellucida ◽  
Cortical Granules ◽  
Hardening Effect

scholarly journals Variations in modifications of sugar residues in hamster zona pellucida after in vivo fertilization and in vitro egg activation

Reproduction ◽  
2002 ◽  
pp. 671-682 ◽  
Author(s):  
M El-Mestrah ◽  
FW Kan
Keyword(s):  
Quantitative Analysis ◽  
Zona Pellucida ◽  
Hydrolytic Enzymes ◽  
Outer Region ◽  
Egg Activation ◽  
Cortical Granules ◽  
Neuraminidase Treatment ◽  
Thin Sections

Lectins were used as probes in conjunction with quantitative analysis to investigate the distribution of different carbohydrate residues in hamster zona pellucida and their possible modification patterns after in vivo fertilization and in vitro egg activation. Several lectins including HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), DBA, AAA and MAA were used to label the zona pellucida of both unfertilized and fertilized eggs. With the exception of PNA and BSAIB(4), the same lectins were also used to label the zona pellucida of oocytes activated in vitro. A multicomparison quantitative analysis of the density of labelling in the inner and outer regions of the zona pellucida before and after fertilization in vivo, as well as after in vitro egg activation, was performed. Of all the lectins studied, preferential localization of labelling by RCA-I and DSA to the inner zona pellucida of unfertilized eggs was observed. After in vivo fertilization, there was an increase in labelling in the inner region of the zona pellucida when thin sections of fertilized oocytes were incubated with HPA, BSAIB(4) and AAA. Although increased labelling by RCA-I was observed, a significant decrease in labelling intensity was obtained with WGA and the sequence Neu-WGA in both the inner and outer zona pellucida of fertilized oocytes. A significant increase in the density of labelling with WGA was also observed after digestion with neuraminidase. In parallel, when hamster oocytes activated in vitro were compared with those fertilized in vivo, a difference in lectin-gold labelling was observed in both the inner and outer region of the zona pellucida. Labelling with HPA, WGA, DSA and MAA increased significantly in both the inner and outer regions of the zona pellucida, whereas labelling by DBA significantly decreased in the inner portion of the zona pellucida. After neuraminidase treatment, a significant increase in labelling density was observed when thin sections of in vitro-activated oocytes were incubated with WGA. These results demonstrate: (i) the post-fertilization modifications of major saccharidic determinants that may play a role in the sperm-egg interaction process of fertilization in vivo; and (ii) that the modified properties of zonae pellucidae of fertilized and in vitro-activated eggs resulting from the action of hydrolytic enzymes, as well as glycoproteins released through exocytosis of cortical granules, are not identical.

227 CALRETICULIN, A 60-kDa PROTEIN, PREVENTS POLYSPERMY IN ZONA PELLUCIDA-FREE PIG OCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 261
Author(s):  
R. Romar ◽  
C. Soriano-Úbeda ◽  
M. D. Saavedra ◽  
J. Gadea ◽  
M. Avilés ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Calcium Binding ◽  
Statistical Significance ◽  
P Value ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Chaperone Protein ◽  
The Mean

After gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. Calreticulin (CRT), a calcium-binding highly conserved protein of 60 kDa, is contained in cortical granules from hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60), and we recently showed it is exocytosed from chemically activated ZP-free pig oocytes (Romar et al. 2012 Reprod. Fertil. Dev. 24). When pig ZP-enclosed oocytes were incubated with CRT, monospermy was not improved (Romar et al. 2011, Maternal communication with gametes and embryo, p. 72), suggesting that the likely role of CRT in preventing polyspermy might be carried out at the oolemma level. Our objective was to evaluate whether CRT prevents polyspermy in pig ZP-free oocytes by treating the cells with this protein before being inseminated. In vitro-matured cumulus–oocyte complexes (44 h, NCSU-37 medium) were decumulated and ZP was digested with Tyrode’s acid. The ZP-free oocytes were incubated for 30 min in TALP medium supplemented with 0, 100, 1000, and 5000 pg of CRT (ab91577, Abcam, Cambridge, MA, USA) per oocyte. After washing, ZP-free oocytes were inseminated (25 000 sperm mL–1) and gametes were co-cultured for 18 h. Putative zygotes were fixed and stained with Hoechst 33342 to analyse the fertilization results. Four replicates with 30 to 35 oocytes per group were done, and results were analysed by one-way ANOVA. A P-value ≤0.05 was taken to denote statistical significance. Incubation with CRT did not affect penetration rates that were similar among groups (77.12 ± 3.88 and 72.73 ± 4.07, respectively, for the 0- and 5000-pg CRT groups). However, the mean number of sperm per penetrated oocyte decreased from 3.01 ± 0.28 (0-pg group) to 2.07 ± 0.16 (5000-pg group), and monospermy rate increased from 30.77 ± 4.87 (0-pg group) to 52.27 ± 5.36 (5000-pg group; P ≤ 0.05). Incubation with CRT did not affect the number of sperm attached to oolemma, which was similar among all groups (11.45 ± 1.16 v. 10.75 ± 1.17, respectively, for 0 and 5000 pg of CRT). These preliminary data suggest that CRT, a protein exocytosed after oocyte activation, participates in the membrane block to polyspermy in pigs. Future studies to describe the exact mechanism of action of this chaperone protein are necessary. Supported by MEC and FEDER (AGL2009-12512-C02-01).

scholarly journals Accelerated modification of the zona pellucida is the primary cause of decreased fertilizability of oocytes in the 129 inbred mouse strain

Zygote ◽  
2010 ◽  
Vol 19 (4) ◽  
pp. 315-322 ◽  
Author(s):  
Toshiaki Hino ◽  
Kanako Oda ◽  
Kenji Nakamura ◽  
Hiroyuki Tateno ◽  
Yutaka Toyoda ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Mouse Strain ◽  
Inbred Mouse ◽  
Inbred Mouse Strain ◽  
Cortical Granules ◽  
Small Litter ◽  
Small Litter Size ◽  
Sperm Plasma Membrane ◽  
High Fertilization

SummaryWe investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.

Mammalian gamete fusion depends on the inhibition of ovastacin by fetuin-B

2014 ◽  
Vol 395 (10) ◽  
pp. 1195-1199 ◽  
Author(s):  
Walter Stöcker ◽  
Konstantin Karmilin ◽  
André Hildebrand ◽  
Hagen Westphal ◽  
Irene Yiallouros ◽  
...  
Keyword(s):  
Plasma Protein ◽  
Zona Pellucida ◽  
Cortical Granules ◽  
Gamete Fusion ◽  
Mammalian Oocyte ◽  
Zona Hardening ◽  
Sperm Penetration ◽  
Cortical Vesicles

Abstract The zona pellucida, a glycoprotein matrix surrounding the mammalian oocyte, hardens after intrusion of the first spermatozoon, thus protecting the embryo until implantation and preventing multiple fertilizations (polyspermy). Definitive zona hardening is mediated by the metalloprotease ovastacin, which is released from cortical granules of the oocyte upon sperm penetration. However, traces of ovastacin seep from unfertilized eggs to cause zona hardening even in the absence of sperm. These small amounts of protease are inactivated by the plasma protein fetuin-B, thus keeping eggs fertilizable. Once a sperm has penetrated the egg, ovastacin from cortical vesicles overrides fetuin-B and initiates zona hardening.

Cytoplasmic maturation of the marsupial oocyte during the periovulatory period

1996 ◽  
Vol 8 (4) ◽  
pp. 509 ◽  
Author(s):  
KE Mate
Keyword(s):  
Zona Pellucida ◽  
Polar Body ◽  
Monodelphis Domestica ◽  
Cortical Granules ◽  
Nuclear Maturation ◽  
Bettongia Penicillata ◽  
Maturation In Vitro ◽  
First Polar Body

During the period immediately before ovulation, the oocytes of most eutherian and marsupial mammals complete the first meiotic maturation division and extrude the first polar body. In marsupials, this phase of nuclear maturation is accompanied by an increase in size of the egg and maturation of cytoplasmic components. Oocytes from at least four marsupial species, Trichosurus vulpecula, Macropus eugenii, Bettongia penicillata and Monodelphis domestica, continue to grow after formation of the follicular antrum and, although the rate of growth slows in larger follicles, it continues into the period immediately before ovulation. The basis of this growth is unknown but may include accumulation of fluid and/or yolk-like material. Maturational changes within the cytoplasm of the oocyte also occur during the periovulatory period, including the accumulation of cortical granules. Differences in the structure of the zona pellucida are also evident between follicular and ovulated eggs; these differences are suggestive of compression of the zona pellucida, but may involve the addition of extra material. These findings suggest that the marsupial oocyte may not achieve complete cytoplasmic maturity until after ovulation; however, their relevance to fertilization and embryonic development require further investigation. Like those of eutherian mammals, marsupial oocytes undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system. It is not known whether the preovulatory cytoplasmic changes seen in marsupial oocytes matured in vivo also occur during maturation in vitro.

scholarly journals Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

2017 ◽  
Vol 45 (1) ◽  
pp. 7
Author(s):  
Joana Claudia Mezzalira ◽  
Lain Uriel Ohlweiler ◽  
Norton Klein ◽  
Daniela Dos Santos Brum ◽  
Fábio Gallas Leivas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Follicular Fluid ◽  
Zona Pellucida ◽  
Antioxidant Properties ◽  
Bovine Embryo ◽  
Cortical Granules ◽  
Embryo Production ◽  
Oocyte Vitrification ◽  
Sperm Penetration ◽  
Vitrification Solution

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).Discussion: Oocytes cryopreserved in TH based solution are known to show certain rigidity in the zona pellucida, being this event a possible cause to spermatozoa penetration disruption. Our results agree with that, since the fertilization rate for TH-Vitri was significantly lower than for the FF-Vitri. In contrast, GV oocytes vitrified in total versus partial FF based solution showed similar maturation and fertilization rates as the Fresh Control, evidencing the beneficial effect of FF during the course of vitrification. It is possible that FF helped to adjust oocyte maturation, allowing a better nuclear-cytoplasmic synchrony. Also, it might have provided some protection due to its antioxidant properties. The releasing of cortical granules induced by freezing, lead to a zona pellucida hardening and failure in sperm penetration. Factors present in the FF might block this premature releasing of cortical granules, thus ensuring that the egg retains its ability to be fertilized after maturation. The blastocysts produced from the FF-Vitri oocytes were the only ones that had the average ICM similar to the Fresh Control, evidencing that besides the similarity in morula + blastocyst rates, the embryos derived from oocytes vitrified in FF solution have also yielded best quality. When vitrified warmed oocytes were submitted to ICSI, there was an increase in the blastocyst production. This increment of embryo production with ICSI evidences a pathway to overcome the zona pellucida biological barrier. In conclusion, the use of FF as base for vitrification solution improves further embryo development; ICSI increases the embryo production of vitrified/warmed bovine GV stage oocytes.

Structural and functional differentiation of follicular and oviductal mouse oocytes visualised with FITC-protein conjugates

Zygote ◽  
1993 ◽  
Vol 1 (4) ◽  
pp. 297-307 ◽  
Author(s):  
Haekwon Kim ◽  
Allen W. Schuetz
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Polar Body ◽  
Ionophore A23187 ◽  
Perivitelline Space ◽  
Cortical Granules ◽  
Functional Changes ◽  
Protein Conjugates ◽  
Mouse Oocytes

SummaryThe fluroscence labelling characteristics of mouse oocytes were examined at various stages of periovulatory differentiation using FITC-protein conjugates. The zona pellucida perivitelline space and plasma membrane underwent visible changes which were developmentally and environmentally related. Following exposure to fluorescein isothiocyanate (FITC)-casein conjugates, the zona pellucida (ZP) of germinal vesicle stage (GV) ovarian oocytes exhibited a bright, amorphous, mesh-like staining pattern (immature type). In contrast, mature polar body stage (PB) oocytes, either ovarian or oviductal, displayed faint, spotty fluorescence labelling of the ZP (mature type). The perivitelline space (PVS) of mature ovarian oocytes (12 h post-hCG) failed to label, whereas approximately 50% of oviductal oocytes showed PVS labelling. The incidence of PVS staining increased with postovulatory age, possibly as a result of the accumulation of materials secreted by the oviduct. Following in vivo or in vitro fertilisation of oocytes, a characteristic pattern of plasma membrane (PM) labelling was observed. Similar patterns of PM labelling were seen in oocytes parthenogenetically activated with ethanol or ionophore (A23187) but not in control oocytes. The pattern of PM labelling observed with FITC-protein conjugates was strikingly similar to that observed with FITC-labelled lectins, which are thought to interact with glycoconjugates released from cortical granules. Immature type of ZP staining also occurred when GV oocytes were treated with FITC alone or with a variety of FITC-protein conjugates. Thus, protein may not be required for labelling of the ZP by FITC-protein conjugates as previously thought. FITCconjugated proteins including casein, bovine serum albumin, peroxidase and non-immune immunoglobulin G (IgG), all labelled the PM of activated oocytes; however, FITC-IgG failed to label the PVS. Results demonstrate for the first time that various components of viable mouse oocytes exhibit and undergo characteristic structural and functional changes during periovulatory differentiation as evidenced by their interaction with one or more FITC-protein conjugates and/or FITC. On the basis of these results the intrafollicular and oviductal mechanisms mediating these changes are discussed as is the possibility that the fluorescent molecule attached to conjugates may play a role in oocyte labelling.

Early Reactions of the Rodent Egg to Spermatozoon Penetration

1956 ◽  
Vol 33 (2) ◽  
pp. 358-365 ◽  
Author(s):  
C. R. AUSTIN ◽  
A. W. H. BRADEN
Keyword(s):  
Zona Pellucida ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Cortical Granules ◽  
Rats And Mice ◽  
Mouse Eggs

In the rat, mouse and hamster the spermatozoon passes rapidly through the thick, homogeneous zona pellucida surrounding the egg and the head almost immediately becomes attached to the surface of the inner cytoplasmic mass or vitellus. As a result of this attachment a block to polyspermy is developed in rat and mouse eggs. In the hamster a block is apparently not formed. It seems likely, therefore, that the disappearance of cortical granules in the hamster egg, also an outcome of contact with the spermatozoon head, could signal the release of an agent that is responsible, after crossing the perivitelline space, for bringing about the zona reaction, reducing the penetrability of the zona pellucida to spermatozoa. Data suggest that this mechanism exists also in rats and mice, although a cortical granule response has not been distinguished in these animals. Thus, attachment of the spermatozoon head to the vitellus probably elicits both the zona reaction and the block to polyspermy. These changes appear to be specific to spermatozoon penetration and to be initiated before the spermatozoon head passes through the surface of the vitellus and before the resumption of the second meiosis.

Precocious loss of cortical granules during mouse oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida

1990 ◽  
Vol 137 (1) ◽  
pp. 46-55 ◽  
Author(s):  
Tom Ducibella ◽  
Shigeaki Kurasawa ◽  
Sathyabhama Rangarajan ◽  
Gregory S. Kopf ◽  
Richard M. Schultz
Keyword(s):  
Zona Pellucida ◽  
Mouse Oocyte ◽  
Meiotic Maturation ◽  
Cortical Granules ◽  
Oocyte Meiotic Maturation
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