Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1,2-propanediol

2013 ◽  
Vol 25 (4) ◽  
pp. 609 ◽  
Author(s):  
Dun-Gao Li ◽  
Yan Zhu ◽  
Feng-Ying Xing ◽  
Shan-Gang Li ◽  
Xue-Jin Chen ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Survival Rates ◽  
Slow Freezing ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Artificial Activation ◽  
Pronuclear Formation

The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation–activation, embryonic development of oocytes–zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5 h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze–thawing did not affect the later development of zygotes.

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection

Zygote ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 111-116 ◽  
Author(s):  
Yasuyuki Araki ◽  
Midori Yoshizawa ◽  
Hiroyuki Abe ◽  
Yoshihiko Murase ◽  
Yasuhisa Araki
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Control Mouse ◽  
Formation Rate ◽  
Human Sperm ◽  
Strontium Chloride ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Sperm Chromosomes ◽  
Artificial Activation ◽  
Infertile Group

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p<0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.

scholarly journals Effects of cryoprotectants and low temperatures on hatching and abnormal embryo development of Prochilodus lineatus (Characiformes: Prochilodontidae)

2017 ◽  
Vol 15 (3) ◽  
Author(s):  
Raphael S. Costa ◽  
Fabrício M. S. de Souza ◽  
José A. Senhorini ◽  
Rosicleire Veríssimo-Silveira ◽  
Alexandre Ninhaus-Silveira
Keyword(s):  
Embryonic Development ◽  
Low Temperatures ◽  
Survival Rates ◽  
Ice Nucleation ◽  
Small Bodies ◽  
Prochilodus Lineatus ◽  
Morphological Alterations ◽  
Membrane Rupture ◽  
Significant Difference ◽  
Chilling Sensitivity

ABSTRACT This study evaluated the effect of the cryoprotectants and the low temperatures on the embryonic development of Prochilodus lineatus, describing their main morphological alterations. On chilling sensitivity test, the survival rates at the twenty somites stage (20S) were 53.6% at 0ºC, and 100% in 5ºC. To test toxicity, the embryos were exposed to a graded series of 1,2-Propanediol (PROP), dimethyl sulfoxide (Me2SO4) and glycerol (GLY), terminating in a solution of high osmolarity. There was no significant difference in the embryos survival of toxicity test between series of PROP and Me2SO4 in the 6S and 20S. In the cooling protocols, were evaluated the effects of low temperature associated with cryoprotectants. At 5ºC, PROP showed survival rates above 75% in the gastrula stage (G) and above 90% in the 6S and 20S stages. High rates of abnormalities were observed, and the most recurrent were: small bodies, fins presenting uncontrolled cell growth, membrane rupture, and retraction. These results demonstrate the need to use cryoprotectant solutions, even when there is no ice nucleation, and, on the other hand, shows that high cryoprotectant concentrations promote numerous morphological lesions, compromising normal embryonic development.

P–109 Comparison between the outcome of sperm vitrification protocol and conventional slow freezing protocol for semen cryopreservation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
K Patel ◽  
N Sharma ◽  
V Mishra ◽  
R Aggarwal ◽  
A Suthar ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Survival Rates ◽  
Semen Sample ◽  
Slow Freezing ◽  
Cellular Damage ◽  
Fragmentation Index ◽  
Significant Difference ◽  
Group 2 ◽  
Dna Fragmentation Index ◽  
Group 1

Abstract Study question Does sperm vitrification technique helps in increasing sperm survival and low DNA fragmentation index post warming. Summary answer Sperm vitrification protocol results in better motility, high progression and low DNA fragmentation index as compared to slow freezing. What is known already Cryopreservation is ceasing and resuming the cell metabolism, which can be achieved by different techniques like slow freezing and vitrification .Vitrification allows solidification of the cells and extracellular milieu into a glass like state without formation of ice which protects intracellular and extracellular ice formation, and further helps in avoiding different types of cryo-injuries and cellular damage. Study design, size, duration: Comparative study from July 2019 to Oct 2020 in IVF unit of IKDRC Hospital. Two hundred and ten patients were randomized by computer generated list and divided into two groups. Group 1 (n = 110) samples cryopreserved by vitrification and Group 2 (n = 100) samples cryopreserved by conventional slow freezing. Participants/materials, setting, methods Semen sample were analyzed by WHO 2010 laboratory manual, including all normozoospermic samples , other abnormal samples were excluded from the study . Method of semen preparation before cryopreservation is similar for both the groups, double density gradient method of preparation was used . Semen sample with high viscosity, hypo and hyper-spermia were also excluded. Similar cryovials of 2ml volume were used for both groups. Main results and the role of chance In group 1 where samples were cryopreserved by vitrification sperm motility was (54.3% vs 49.2%)vs in group 2 where samples were cryopreserved by slow freezing , non- significant difference were observed , but progressive motility was significantly higher in group 1 as compared to group 2 (36.8%vs17.9%) and DNA fragmentation index is significantly lower in group 1 vitrification than in group 2slow freezing ( 9.7% vs 20%). Limitations, reasons for caution Technical proficiency of the operator to avoid human errors and still larger randomized control studies are needed to strengthen these results Wider implications of the findings: Our study demonstrates that vitrification is better than slow freezing of human sperm, improved survival rates with high progression were found with vitrification and low DNA fragmentation index were also observed in samples cryopreserved with vitrification protocol. Trial registration number Not applicable

Microinjection of mouse phospholipase Cζ complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development

2008 ◽  
Vol 20 (8) ◽  
pp. 875 ◽  
Author(s):  
Sylvia J. Bedford-Guaus ◽  
Sook-Young Yoon ◽  
Rafael A. Fissore ◽  
Young-Ho Choi ◽  
Katrin Hinrichs
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Assisted Reproduction Technologies ◽  
Significant Difference ◽  
Developmental Rates ◽  
Advanced Stages ◽  
Consistent Method

Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Cζ (PLCζ) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca2+]i oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCζ complementary (c) RNA (1, 0.5 or 0.25 μg μL–1) into mare oocytes. The frequency of [Ca2+]i oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 μg μL–1 PLCζ cRNA (41.1 ± 5.3, 47 ± 4.0 and 55.4 ± 9.0, respectively). However, [Ca2+]i oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 μg μL–1 PLCζ cRNA (570.7 ± 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCζ (P > 0.05; range 97–100%), but the proportion of oocytes reaching advanced stages of embryonic development (>64 nuclei) was significantly lower for oocytes injected with 0.25 μg μL–1 PLCζ cRNA (3%) than for those injected with 1 μg μL–1 PLCζ cRNA (15%). Based on these results, microinjection of PLCζ may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca2+]i signalling in this species.

Comparison between different combinations of chemical treatment on parthenogenetic activation of mouse oocytes and its subsequent embryonic development

2013 ◽  
Vol 17 (3) ◽  
pp. 196-202 ◽  
Author(s):  
Siti Khadijah Idris ◽  
Ramli Bin Abdullah ◽  
Wan Khadijah Wan Embong ◽  
Mohammad Mijanur Rahman
Keyword(s):  
Embryonic Development ◽  
Chemical Treatment ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection

2014 ◽  
Vol 26 (6) ◽  
pp. 847 ◽  
Author(s):  
M. E. Arias ◽  
R. Sánchez ◽  
J. Risopatrón ◽  
L. Pérez ◽  
R. Felmer
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Membrane Integrity ◽  
Control Group ◽  
Developmental Potential ◽  
Bovine Spermatozoa ◽  
Pronuclear Formation ◽  
First Time ◽  
In Vitro Embryonic Development

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10–50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

234 PARTHENOGENETIC ACTIVATION OF DOMESTIC CAT OOCYTES USING STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 196 ◽  
Author(s):  
C. Wang ◽  
K. Lee ◽  
S. Koh ◽  
Z. Machaty
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Blastocyst Formation ◽  
Formidable Challenge ◽  
Significant Difference

Cloning domestic cats is useful in comparative medicine programs as it may provide insight into unique disease mechanisms and facilitate investigation of new therapeutic options. It is also believed to be beneficial for the conservation of precious animal models. However, as in many species, low birth rates after nuclear transfer remain a formidable challenge. One potential reason for the low efficiency is poor embryo development following activation of the reconstructed oocytes. The number of methods available to induce a transient increase in the oocytes' cytosolic free calcium level to stimulate development is rather limited. Although strontium has been reported to successfully activate the developmental program of mature mouse and rat oocytes, it was without effect in all other species studied. Here we investigated the effect of strontium on mature cat oocytes. Oocytes collected from the cat ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mm cysteine, 0.1 mm cysteamine, 1 IU mL–1 eCG, 2 IU mL–1 hCG, 25 ng mL–1 epidermal growth factor (EGF) for 24 h. For intracellular calcium measurements, mature oocytes were incubated in the presence of 2 µ m fura-2Am, a calcium indicator dye, and 0.02% pluronic F-127 for 40 min. Individual oocytes were transferred into calcium-free HEPES, and SrCl2 was added to the medium at a final concentration of 20 mm. Changes in the intracellular free calcium levels were then monitored using an InCyt Im2™ fluorescence imaging system (Intracellular Imaging, Cincinnati, OH, USA). Preimplantation embryonic development was also evaluated by incubating the oocytes with 20 mm SrCl2 in calcium-free HEPES medium supplemented with 7.5 µg mL–1 cytochalasin B for 6 h. Control oocytes were activated by two 20-µs-long, 100 kV cm–1 direct current pulses and incubated in the presence of 7.5 µg mL–1 cytochalasin B for 6 h. After activation, the oocytes were cultured in FOCM for 6 days. At the end of the culture period, embryonic development was recorded; the nuclear number of the embryos was also determined after staining with Hoechst 33342. Data were subjected to one-way ANOVA, and differences between treatments were analyzed using the Tukey test. We found that strontium triggered a transient rise in the intracellular free calcium concentration in all oocytes tested (N = 20). Strontium treatment also induced cleavage in 49.7% (92/185) of the oocytes, while 4.9% (9/185) of the activated oocytes developed to the blastocyst stage. In the electroporated group, cleavage frequency was 57.1% (104/182) and blastocyst formation was 8.8% (16/182). Data analysis showed that there was no significant difference between the two groups in terms of cleavage frequency and blastocyst formation. This is the first study to demonstrate that strontium can induce cytoplasmic calcium increase in cat oocytes and trigger development up to the blastocyst stage. The results also indicate that SrCl2 may be useful for oocyte activation during cat nuclear transfer. Additional studies are needed to determine whether SrCl2 can trigger development more effectively than current activation techniques.

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 299-307 ◽  
Author(s):  
Masatsugu Asada ◽  
Hong Wei ◽  
Rie Nagayama ◽  
Masafumi Tetsuka ◽  
Hajime Ishikawa ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Subsequent Development ◽  
Minke Whale ◽  
Oocyte Activation ◽  
Minke Whales ◽  
Maturation Medium ◽  
Significant Difference ◽  
Serum Gonadotropin ◽  
Pronuclear Formation

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17β(E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.

Production of inbred offspring by intracytoplasmic sperm injection of oocytes from juvenile female mice

2018 ◽  
Vol 30 (3) ◽  
pp. 451 ◽  
Author(s):  
Jie Zhu ◽  
Wei Cui ◽  
Yan-Feng Dai
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Mouse Strains ◽  
Control Group ◽  
Juvenile Female ◽  
Female Mice ◽  
Adult Mice ◽  
Significant Difference ◽  
Pronuclear Formation

The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P < 0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10 mM SrCl2 + 5 μg mL−1 cytochalasin B for 5–6 h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P > 0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6 h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P > 0.05). Oocytes from juvenile mice activated in 10 mM SrCl2 for 2 h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P < 0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.

scholarly journals Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa

Reproduction ◽  
2003 ◽  
pp. 489-499 ◽  
Author(s):  
SJ Bedford ◽  
M Kurokawa ◽  
K Hinrichs ◽  
RA Fissore
Keyword(s):  
Embryonic Development ◽  
Intracellular Calcium ◽  
Intracytoplasmic Sperm Injection ◽  
Animal Species ◽  
Mammalian Species ◽  
Calcium Oscillations ◽  
Large Animal ◽  
Oocyte Activation ◽  
Mouse Oocytes ◽  
Species Specific

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

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