scholarly journals 313 EFFECTS OF ETHANOL TREATMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) ON SPERM AFTER FORMATION AND THE MICROTUBULE ORGANIZATION OF BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 307
Author(s):  
N. Fujinami ◽  
Y. Hosoi ◽  
H. Kato ◽  
T. Mitani ◽  
K. Matsumoto ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Formation Rate ◽  
Ethanol Treatment ◽  
Microtubule Organizing Center ◽  
Cleavage Rate ◽  
Microtubule Organization ◽  
Male And Female ◽  
Bovine Oocytes ◽  
Sperm Aster

The cleavage rate of bovine embryos is very low without activation of oocytes after intracytoplasmic sperm injection (ICSI), although both male and female pronuclei are formed. We previously reported that the stimulus due to the injected sperm alone was sufficient to lower the MPF activity of bovine oocytes after ICSI, and the activation treatment of oocytes with ethanol at 4 h after ICSI served to maintain the low levels of MPF activity until the next cell cycle started (Fujinami et al. 2004 J. Reprod. Dev. 50, 171–178). These results suggested that activation treatment is necessary to improve the embryonic development after bovine ICSI. In bovine fertilization, the sperm introduces the centrosome into the oocyte. The centrosome acts as the microtubule-organizing center and microtubules are organized within the oocyte. It is reported that the sperm aster is important for the normal fertilization process. Therefore, failure of sperm aster formation possibly causes the failure of cleavage following fertilization. To investigate the reason of the low cleavage rate after bovine ICSI without artificial activation treatment, we examined sperm aster formation and the microtubule organization in bovine oocytes with or without activation treatment after ICSI. Bull spermatozoa immobilized by piezopulse was injected into bovine oocytes matured in vitro. At 4 h after ICSI, oocytes were treated with 7% ethanol in TCM199 for 5 min for activation. Oocytes were fixed at 6 and 12 h after ICSI, and the microtubule organization was examined by using specific antibodies and immunofluorescence microscopy. The cleavage rate (51% vs. 15%) and the developmental rate to the blastocyst stage (13% vs. 3%) were increased by ethanol treatment after ICSI (with or without ethanol treatment, respectively, P < 0.05). In oocytes activated with ethanol after ICSI, both the sperm aster formation rate at 6 h and the microtubule organization rate at 12 h after ICSI were significantly higher than in oocytes without activation treatment (58%, 80% vs. 12%, 26%, P < 0.05). It was reported that the sperm aster has an important role for the pronuclear movement to make the male and female pronuclei come into close apposition. From these results, it was concluded that oocyte activation after bovine ICSI promoted sperm aster formation and microtubule organization, and was effective to improve embryonic development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

230 ACTIVATION OF IN VITRO-MATURED BOVINE OOCYTES WITH IONOMYCIN AND ROSCOVITINE AFTER INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 194
Author(s):  
C. B. Fernandes ◽  
L. G. Devito ◽  
L. R. Martins ◽  
T. S. Rascado ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Sperm Concentration ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Sperm Suspension ◽  
Artificial Activation ◽  
Bovine Oocytes

In all mammalian species studied so far, fertilization induces oocyte activation necessary for pronuclear formation, syngamy, and the beginning of embryonic cleavage. The aim of this experiment was to evaluate the effectiveness of a protocol for artificial activation for bovine oocytes using ionomycin and roscovitine either in combination with intracytoplasmic sperm injection (ICSI) or alone. In this study, ionomycin was used to facilitate the increase of intracellular calcium, due to the release of calcium from intracellular stores. This compound was used in conjuction with roscovitine, a specific cdc2 kinase inhibitor. The success of the treatment was compared with that of oocytes fertilized by IVF. Three replicates were carried out using bovine oocytes harvested from slaughterhouse ovaries. In vitro-matured oocytes were cultured in TCM-199 plus 10% FCS, pyruvate, estradiol, hCG, and gentamicin at 39�C in an atmosphere of 5% of CO2 in air for 20 h. After in vitro maturation, oocytes were divided into 3 groups. For parthenogenetic activation, 100 oocytes were stripped of cumulus cells and placed in H-MEM plus 10% FCS and 5 µm ionomycin for 8 min, maintained in H-MEM plus 10% FCS, 66 mm roscovitine and 7.5 mg mL–1 cytochalasin B for 6 h, and placed into culture. In the ICSI group, oocytes were denuded and transferred to 5-µL H-MEM plus 20% FCS drops. Only MII oocytes were microinjected. The sperm drop was prepared with a mixture of 4 µL polyvinylpyrrolidone (PVP) and 1 µL of the sperm suspension produced by Percoll gradient. For injection, a single normal mobile sperm was aspirated with the tail first. A single oocyte was fixed by holding the pipette to position the polar body at the 6 or 12 o'clock position. The injection pipette was pushed through the zona pellucida and the oolema and the spermatozoan was released into the cytoplasm. After ICSI, the oocytes were subjected to the same activation protocol described earlier and cultured. For IVF, sperm was prepared by swim-up and 100 oocytes were fertilized in Fert-Talp for 18 h (sperm concentration: 1 � 106). All oocytes were cultured in HTF:BME plus 0.6% BSA, 10% FCS, 0.01% myoinositol, and gentamycin at 39�C in an atmosphere of 5% of CO2 in air for 72 h. Cleavage was evaluated visually and the embryos were stained with Hoechst 33342 for estimation of nuclei numbers. The data were analyzed by ANOVA, followed by the Tukey test (P < 0.05). The results showed a cleavage rate of 76% for the IVF group, 57% for the ICSI group, and 51% for the parthenogenic group. The artificial activation proposed was efficient in inducing oocyte activation and cleavage; however, the rates obtained were significantly lower then the ones observed after IVF. Injection of a viable sperm into the oocyte through ICSI did not improve the cleavage rate after activation. This result indicates that the membrane fusion and/or sperm interaction with the oocyte during fertilization is important for the physiological modifications that result in oocyte cleavage in bovine.

scholarly journals Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 453-463
Author(s):  
Joao Alveiro Alvarado Rincón ◽  
Patricia Carvalho Gindri ◽  
Bruna Mion ◽  
Ferronato Giuliana de Ávila ◽  
Antônio Amaral Barbosa ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Lps Challenge ◽  
Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

The effects of in vitro maturation duration on maturation rate and intracytoplasmic sperm injection-derived embryonic development in goats

2015 ◽  
Vol 49 (6) ◽  
Author(s):  
A. H. Nor Farizah ◽  
M. M. Rahman ◽  
W. E. Wan Khadijah ◽  
R. B. Abdullah
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Cumulus Cell ◽  
Cleavage Rate ◽  
Maturation Rate ◽  
Cell Layers ◽  
Embryo Cleavage

The aim of the present study was to evaluate the effects of <italic>in vitro</italic> maturation (IVM) duration on maturation rate and intracytoplasmic sperm injection (ICSI)-derived embryonic development in goat embryos. Donor goats were superovulated following oestrus synchronisation prior to laparoscopic oocyte pick-up. The quality of oocytes was scored based on cumulus cell layers, which were graded A, B and C. The oocytes were cultured in IVM medium with two different IVM durations (18-21 h and 22-25 h) for the ICSI procedure. A total of 327 matured oocytes were used, of which 157 and 170 oocytes were used for 18-21 h and 22-25 h IVM duration, respectively. The embryo cleavage rate of Grade A from the 18-21 h IVM group was significantly (P<0.05) higher than that of grades B and C. However, in the 22-25 h IVM duration group, the cleavage rates for all grades of oocytes were not significantly (P>0.05) different. Regardless of oocyte grades, no significant differences in maturation rates and cleavage rates for all stages of embryonic development between the two groups of IVM durations were observed. The results suggest that both IVM durations have the same potential in ICSI-derived embryonic development.

382 SYNCHRONIZING ACTIVATION TIME WITH MAXIMUM MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE LEVELS DOES NOT IMPROVE BOVINE INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 306
Author(s):  
B. J. Woodward ◽  
W. E. Maalouf ◽  
K. H. S. Campbell
Keyword(s):  
Protein Kinase ◽  
Intracytoplasmic Sperm Injection ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cleavage Rate ◽  
Mitogen Activated Protein ◽  
Activation Times ◽  
Bovine Oocytes ◽  
Injection Procedure ◽  
External Activation

The success of bovine intracytoplasmic sperm injection (ICSI) remains low because of the tightly packed sperm nucleus failing to decondense and the need for additional external activation. Maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) assist the breakdown of the nuclear envelope. Activities are high at the early metaphase II (MII) stage but decrease with time. We assessed MPF and MAPK dynamics during the MII stage from 24 h postmaturation (hpm) onward. We then tested the hypothesis that ICSI may be more successful if the external activation is performed following sperm exposure to maximum MPF and MAPK levels. Bovine oocytes were matured in M199 with 10% FCS, 5 µg mL−1 FSH, 5 µg mL−1 LH, 1 µg mL−1 estradiol, and 50 µL mL−1 gentamycin, and cumulus cells were removed at 18 hpm. Groups of 10 MII oocytes were sampled from 24 hpm and analyzed for MPF and MAPK activities as previously described (Ye et al. 2003 Reproduction 125, 645–656). For ICSI, MII oocytes were moved to HSOF supplemented with amino acids and BSA (HSOFaa) and injected with sperm from 19 to 22 hpm. Further MII oocytes were subjected to sham or no injections at this time. Injections were performed manually using a 1480-nm diode laser (XY-clone; Hamilton-Thorne, Beverly, MA, USA) to breach the zona pellucida. Because the maximum kinase activities were reached at 28 hpm, oocytes were activated in 2 groups: at either 24 or 28 hpm, using HSOFaa containing 7% ethanol followed by culture in mSOFaa with 10 µg mL−1 cycloheximide for 6 h, and then transferred to mSOFaa medium until cleavage assessment on Day 2. Differences in the percentage of cleaved oocytes in the different groups were analyzed by a chi-squared test. From 24 to 28 hpm, there was an increase in both MPF (24.6%) and MAPK (58.7%) levels, followed by a slow decline. There were no significant differences in cleavage rates for sham-injected or control oocytes at the different activation times (P &lt; 0.05). However, a significantly higher cleavage rate was observed following ICSI with activation at 24 hpm compared with 28 hpm (P &lt; 0.001), even though kinase levels appeared maximal at 28 hpm. The manual injection procedure appeared detrimental, because oocytes in the control group showed a significantly higher cleavage rate than sham-injected oocytes (P &lt; 0.01). In conclusion, bovine oocytes subjected to ICSI showed a better cleavage rate if external activation was performed at 24 hpm rather than at 28 hpm, even though the MPF and MAPK levels were maximal at 28 hpm (P &lt; 0.001), even though kinase levels appeared maximal at 28 hpm. The manual injection procedure appeared detrimental, because oocytes in the control group showed a significantly higher cleavage rate than sham-injected oocytes (P &lt; 0.01). In conclusion, bovine oocytes subjected to ICSI showed a better cleavage rate if external activation was performed at 24 hpm rather than at 28 hpm, even though the MPF and MAPK levels were maximal at 28 hpm. Table 1. Percentage of Day 2 cleaved oocytes after intracytoplasmic sperm injection, sham or no injection at 2 different activation times

69 Cloned bovine embryonic development derived from interferon tau knockout cells

2019 ◽  
Vol 31 (1) ◽  
pp. 160 ◽  
Author(s):  
K.-M. Kim ◽  
S.-J. Lee ◽  
S.-Y. Yum ◽  
H.-S. Kim ◽  
H.-J. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Formation Rate ◽  
Development Program ◽  
Donor Cell ◽  
Type I ◽  
Cleavage Rate ◽  
Interferon Tau ◽  
Signal Molecule ◽  
Significant Difference

Interferon tau (IFNT), a type I interferon, is known as a key signal molecule during pregnancy in ruminants because of the necessity in maternal recognition of pregnancy. It is produced in trophectoderm cells of the elongation bovine conceptus at Day 13-21, and peak production is at Day 15 to 17 of pregnancy. In addition, other studies show that it can affect embryonic development and quality. In this study, the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. To proceed with this experiment, immature oocytes from ovaries at a local slaughterhouse were matured in vitro for 22h. To prepare the donor cell with IFNT knockout, somatic cells were transfected with Cas9 and single guide RNA targeting IFNT, and several single derived colonies with high proliferation were isolated for mutation assay. Finally, one colony that had mono-allelic mutation (4bp deletion) was selected and used as the donor cell for SCNT. A donor cell was injected into an enucleated oocyte. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation, and cultured for 7 days in chemically defined medium. For this study, control (n=94) and IFNT knockout groups (n=140) were compared with 4 replications. The results showed no significant difference between control and IFNT knockout groups not only in cleavage rate, but also in blastocyst formation rate (control: 15.7±8.3%, IFNT knockout group: 26.3±13.1%). In addition, the number of blastocyst cell was not different between control (88.2±27.0) and IFNT knockout group (88.0±21.1). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT, and all samples were positive for mutation. In conclusion, these data demonstrated that the disruption of IFNT did not affect embryonic development. In future study, we would transfer these embryos and check the effect of IFNT during pregnancy status. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes

Zygote ◽  
2000 ◽  
Vol 8 (3) ◽  
pp. 267-274 ◽  
Author(s):  
Hong Wei ◽  
Yutaka Fukui
Keyword(s):  
Polar Body ◽  
Formation Rate ◽  
Sperm Head ◽  
Minke Whale ◽  
Balaenoptera Acutorostrata ◽  
Male And Female ◽  
The Mean ◽  
Pronuclear Formation ◽  
Bovine Oocytes

This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 °C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 μm and 28.3 μm vs 22.4 μm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 μm vs 24.7 μm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.

Quantitative ultrastructural reconstruction of small regions within large volumes by correlative light and high voltage electron microscopy of serial 0.25-0.50 μm sections

1987 ◽  
Vol 45 ◽  
pp. 578-581
Author(s):  
Conly L. Rieder ◽  
Frederick J. Miller ◽  
Edwin Davison ◽  
Samuel S. Bowser ◽  
Kirsten Lewis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Mitotic Spindle ◽  
Meiotic Spindle ◽  
High Voltage Electron Microscopy ◽  
Male And Female ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Differential Stability ◽  
Sperm Aster

In this abstract we Illustrate how same-section correlative light and high voltage electron microscopy (HVEM) of serial 0.25-0.50-μm sections can answer questions which are difficult to approach by EM of 60-100 nm sections.Starfish (Pisaster and Asterlas) eggs are fertilized at meiosis I when the oocyte contains two maternal centrosomes (e.g., asters) which form the poles of the first meiotic spindle. Immediately after fertilization a sperm aster is assembled in the vicinity of the male pronucleus and persists throughout meiosis. At syngamy the sperm aster splits to form the poles of the first mitotic spindle. During this time the functional and replicative properties of the maternal centrosome, inherited from the last meiotic division, are lost. The basis for this differential stability, of male and female centrosomes in the same cytoplasm, is a mystery.

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