scholarly journals Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3313-3323 ◽  
Author(s):  
G.D. Moore ◽  
T. Ayabe ◽  
P.E. Visconti ◽  
R.M. Schultz ◽  
G.S. Kopf
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Zona Pellucida ◽  
Polar Body ◽  
Active Form ◽  
Dominant Negative ◽  
Egg Activation ◽  
Cell Stage ◽  
Second Polar Body ◽  
Mouse Eggs

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

Timing of the First Cleavage Division of Haploid Mouse Eggs, and the Duration of its Component Stages

1973 ◽  
Vol 13 (2) ◽  
pp. 553-566 ◽  
Author(s):  
M. H. KAUFMAN
Keyword(s):  
Polar Body ◽  
Time Lapse ◽  
Follicle Cells ◽  
Cleavage Division ◽  
Cell Stage ◽  
Cell Embryo ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mouse Eggs

Mouse eggs were activated by treatment with hyaluronidase which removed the follicle cells, followed by culture in vitro, and examined at the first cleavage mitosis. Second polar body extrusion usually occurred and haploid parthenogenesis was initiated. Air-dried chromosome preparations were made between 11 and 15.5 h after activation. Out of the 308 eggs examined 74 had already progressed to the 2-cell stage; the remaining 234 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase, metaphase (early or ‘pre-chromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after activation. Similar eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronucleus to the beginning of telophase of the first cleavage division was determined. The results of timing studies on the haploid eggs were compared with results obtained from similar observations on the first cleavage division of fertilized eggs which would of course normally be diploid. Artificially activated eggs with 2 pronuclei, resulting from second polar body suppression, were also examined, and serial chromosome preparations during mitosis showed that the 2 pronuclear chromosome groups unite on the first cleavage spindle and divide to give a hetero-zygous diploid 2-cell embryo.

Effects of extremely low osmolarity on fertilized mouse eggs

Development ◽  
1977 ◽  
Vol 37 (1) ◽  
pp. 65-77
Author(s):  
Jolanta Opas
Keyword(s):  
Water Treatment ◽  
Zona Pellucida ◽  
Polar Body ◽  
Preimplantation Development ◽  
Distilled Water ◽  
Second Polar Body ◽  
Mouse Eggs

When fertilized one-cell eggs are subjected to distilled water treatment for 2–6 min, cytoplasm bulges through the sperm-slit in the zona pellucida and forms a cytoplasmic fragment (CF). CFs were observed in 86·5 % of eggs; in 20·9 % of cases CFs contained a pronucleus (or pronuclei). In 53·4 % of eggs permanent incorporation of the second polar body (2 P.B.) into the egg cytoplasm occurred. These phenomena occurring in different combinations produced 6·2 % of haploid eggs, 10·3 % of diploid eggs with a pronucleus replaced by 2 P.B. nucleus, and 43·1 % of triploid eggs. 4·4 % t of eggs were enucleated. The remaining group comprised diploid eggs which were either not affected by the treatment (6·4 %) or lost a certain amount of cytoplasm by formation of an anucleate CF (29·6%). The frequencies of the types of reaction were related to the post-fertilization stage of eggs. All eggs except the enucleated ones were able to develop to the stage of morula or blastocyst. Triploids developed until the 12th day of pregnancy and diploids that had lost up to 15 % of the cytoplasm developed to term. There was a twofold reduction in the percentage of preimplantation development when treated eggs originated from induced rather than spontaneous ovulation.

The early development of haploid and aneuploid parthenogenetic embryos

Development ◽  
1975 ◽  
Vol 34 (3) ◽  
pp. 645-655
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Early Development ◽  
Polar Body ◽  
Chromosome Counts ◽  
Cell Stage ◽  
Haploid Chromosome Number ◽  
Stage Of Development ◽  
Morula Stage ◽  
Second Polar Body ◽  
Similar Development ◽  
Parthenogenetic Embryos

The early development of parthenogenetically activated oocytes has been studied in C57BL × CBA-T6T6 (F1T6) translocation heterozygote mice and C57BL × CBA-LAC (F1LAC) mice. All F1T6 oocytes had either a quadrivalent or a univalent-trivalent configuration at meiosis I; no such chromosome configurations were observed in the F1LAC oocytes. At ovulation 36·5 % of the F1T6 oocytes had 19 or 21 chromosomes, whereas 97 % of the F1LAC had the normal haploid chromosome number of 20. After parthenogenetic activation, chromosome counts at metaphase of the first cleavage mitosis were made of the eggs with a single pronucleus following extrusion of the second polar body. These activated eggs had similar frequencies of 19, 20 and 21 chromosomes as had the oocytes at ovulation. The activated 1-cell eggs were transferred to the oviducts of pseudopregnant recipients and the embryos recovered 3 days later. At this stage of development, most of the F1T6 embryos with 19 chromosomes were no longer found, but the frequency of 21-chromosome embryos was similar to the frequency of 21-chromosome oocytes and activated eggs. There was a similar mean number of cells in the embryos with 20 and 21 chromosomes. The results indicate that nearly all the embryos with 19 chromosomes failed to develop, probably beyond the 2-cell stage, whereas oocytes with 21 chromosomes had a similar development to oocytes with 20 chromosomes up to the morula stage.

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling

Development ◽  
2001 ◽  
Vol 128 (5) ◽  
pp. 645-654
Author(s):  
X. Shi ◽  
S. Amindari ◽  
K. Paruchuru ◽  
D. Skalla ◽  
H. Burkin ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Cytoplasmic Domain ◽  
Protein Activation ◽  
Acrosomal Exocytosis ◽  
G Protein Activation ◽  
Galactosyltransferase I

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.

Fertilisation in the Rabbit

1932 ◽  
Vol 9 (4) ◽  
pp. 403-408
Author(s):  
G. PINCUS ◽  
E. V. ENZMANN
Keyword(s):  
Zona Pellucida ◽  
Critical Period ◽  
Polar Body ◽  
Follicle Cells ◽  
Fallopian Tubes ◽  
Sperm Penetration ◽  
Pass Through ◽  
Second Polar Body

The series of events occurring in the Fallopian tubes of rabbit does mated to fertile bucks may be summarised as follows: The ova liberated from the ovaries and surrounded by the follicle cells become massed together. Sperm penetrate the massed follicle cells, which fall away as the sperm pass through them. At from 1½-3 hours after ovulation the spermatozoa reach the egg. A number of spermatozoa pass through the zona pellucida, but only one, apparently, enters the egg. At the time of sperm penetration the egg shrinks slightly but definitely. The second polar body is given off 45 min. or longer after sperm penetration. The pronuclei are formed after the formation of the second polar body and, at the earliest, 3 hours after ovulation. The critical period for sperm penetration appears to occur at 2-3 hours after ovulation (cf. Pincus,1930).

120 EMBRYONIC AND POSTNATAL DEVELOPMENT OF DIPLOID - TRIPLOID MOUSE CHIMAERAS

2005 ◽  
Vol 17 (2) ◽  
pp. 210
Author(s):  
A. Suwinska ◽  
M. Waksmundzka ◽  
W. Ozdzenski ◽  
A.K. Tarkowski
Keyword(s):  
Postnatal Development ◽  
Polar Body ◽  
Experimental Production ◽  
Cell Stage ◽  
Embryonic Tissues ◽  
Glucose Phosphate ◽  
Successful Attempt ◽  
Second Polar Body ◽  
Painting Probes ◽  
Insight Into

A chimaera is an organism composed of cells derived from two (or more) zygotes. Spontaneously originated diploid-triploid (2n-3n) chimaeric embryos and adults have been described in many species of mammals. In man, between 1960 and 2002 over 30 cases of chimaerism were discovered (van de Laar I et al. 2002 Clin. Genet. 62(5), 376–382). A deeper insight into the developmental consequences of this rare and odd phenomenon requires experimental production of 2n-3n embryos and animals. The present study is the first and successful attempt to produce diploid-triploid chimaeric embryos, fetuses, and postnatal animals in the mouse. Diploid embryos originated from BAMIZ females crossed with BAMIZ males. The zygotes that were the source of triploid embryos were obtained from females F1 (C57Bl/6 × CBA/H) crossed with F1 males as a result of “delayed mating.” The triploidy was induced by suppression of the extrusion of the second polar body with cytochalasin D (1 μg mL−1, 5 h). Diploid-triploid chimaeric embryos were created by aggregation of diploid embryos with triploid embryos at 4–8 cell stage. In chimaeras created according to this procedure, the triploid component was agouti and produced the 1B1B isoform of glucose phosphate isomerase (GPI) and the diploid component was albino and produced the GPI-1A1A isoform. Electrophoresis of GPI was performed in order to determine the contribution of both populations of cells in tissues of embryos and individuals. Over a thousand oocytes were subjected to triploidization. A total number of 201 diploid-triploid aggregates developed into blastocysts and were transplanted to the oviducts of 30 recipients. Our experiment yielded 23 living and 6 dead embryos (age: 8th–19th day) out of which 22 proved to be chimaeric and 3 were adults. Two of these animals were albino but had the triploid component in several internal tissues; both were fertile. The third animal, a male, was an overt chimaera. It turned out to be infertile (no sperm in the ejaculate; testes small and deprived of germ cells). The infertility of this individual is puzzling because the FISH studies with the help of X and Y chromosome painting probes proved that the diploid component was XY and the triploid component was XXX. The results of our study indicate that the rate of postimplantation development of 2n-3n chimaeric embryos is normal or only slightly retarded. Developmental stage of chimaeric embryos was assessed by comparison of their external morphology with normal diploid embryos of equivalent post-coital age according to the descriptions given by Theiler K (1972 The House Mouse, Springer-Verlag, Berlin). With the exception of one embryo lacking both eyes (but otherwise looking quite normal) no other morphological abnormalities were observed. Comparison of the contribution of both components to the fetal and extra-embryonic tissues at the consecutive foetal stages has shown that participation of triploid cells slightly but steadily decreased in all tissues examined. However, the presence of triploid cells in mouse chimaeras was compatible with their normal postnatal development to adulthood.

scholarly journals Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

2018 ◽  
Vol 14 (4) ◽  
pp. 512-514
Author(s):  
Nor Shahida Abdul Rahman ◽  
Mimi Sophia Sarbandi ◽  
Wan Hafizah Wan Jusof ◽  
Zolkapli Eshak ◽  
Salina Othman ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Close Association ◽  
Mitochondrial Fission ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Stage ◽  
Second Polar Body

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development. 

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

39 CLONAL OFFSPRING DERIVED FROM SEPARATED BLASTOMERES IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)

2008 ◽  
Vol 20 (1) ◽  
pp. 100
Author(s):  
C. Iwatani ◽  
J. Okahara-Narita ◽  
J. Yamasaki ◽  
H. Tsuchiya ◽  
R. Torii
Keyword(s):  
Cell Lines ◽  
Zona Pellucida ◽  
Cynomolgus Monkey ◽  
Behavioral Science ◽  
Polar Body ◽  
Calf Serum ◽  
Es Cell ◽  
Cell Stage ◽  
Splitting Technique

There are no reports of cloning by embryo splitting in the cynomolgus monkey, but production of genetically identical monkeys would have tremendous implications for biomedical research, especially for immunological studies, production of disease models, and behavioral science. Cloning would also reduce the number of animals required for the above research by increasing experimental reproducibility. In this study, we tried to produce cynomolgus monkey offspring by embryo splitting and embryo transfer. Controlled ovarian stimulation and oocyte recovery have been previously described by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection. Single spermatozoa were individually immobilized by scoring their tails and picking them up with the injection pipette. The denuded oocyte was held by the holding pipette with the polar body in the 12 o'clock position. The injection pipette was then inserted at the 3 o'clock position and was introduced into the cytoplasm, breaking the ooplasmic membrane by pulling gently. One spermatozoon was injected into the cytoplasm. The injected oocytes were cultured at 38�C in 5% CO2, 5% O2 and 90% N2 in CMRL-1066 medium (Invitrogen, Grand Island, NY, USA) containing 20% calf serum (CS, Invitrogen) for 2–3 days. Splitting was performed using 4- to 7-cell-stage embryos. The zona pellucida was disrupted with acidic Tyrode's solution, and individual blastomeres were separated from the zona-free embryos by 0.25% trypsin-EDTA with added CaCl2 (<1 min). After transferring the zona-free embryos into TALP-HEPES medium, blastomeres were dissociated by pipetting with a 40–50 µm micropipette 4–5 times. These blastomeres were then transferred into empty zonae that had been produced from immature oocytes by the aspiration of ooplasm with a micromanipulator. Sixteen embryos underwent blastomere separation and a total of 33 split embryos were produced. After being cultured for 2–3 h in CMRL-1066 medium containing 20% CS, 30 of these split embryos, comprising 1–4 blastomeres each, were transferred into the oviducts of 23 fertile surrogate mothers at 0 to 5 days after ovulation. Pregnancy was confirmed in two animals (8.7%; 2/23) by ultrasound approximately 30 days after transfer. The pregnancies were uneventful and two normal healthy babies were born without any assistance 159 days after transfer. The low pregnancy rate could be due to the presence of fewer cells in the smaller split embryos, the ruptured zona pellucida, or the in vitro micromanipulation of embryos during blastomere separation and reconstruction. Here we report the first production of viable cloned offspring produced by blastomere separation in the cynomolgus monkey. Since we have previously succeeded in establishing ES cell lines from isolated blastomeres, in the future we will be able to produce genetically identical monkeys from a single 4- to 8-cell-stage embryo using those ES cell lines and the embryo splitting technique.

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