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.

276 ACTIVATION OF ROUND SPERMATID-INJECTED OOCYTES USING PHOSPHOLIPASE C ZETA IN PIGS

2013 ◽  
Vol 25 (1) ◽  
pp. 286
Author(s):  
J. Ito ◽  
E. Yuhara ◽  
A. Nakamura ◽  
N. Kashiwazaki
Keyword(s):  
Phospholipase C ◽  
Mammalian Species ◽  
Treated Group ◽  
Round Spermatid ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Domestic Species ◽  
Electrical Pulses ◽  
Group 2 ◽  
Pronuclear Formation

In several mammalian species, the generation of offspring by round spermatid injection has been reported. However, in domestic species, including pigs, no one has reported success to date. One of the reasons is that round spermatid-injected oocytes require artificial stimuli for oocyte activation, but the developmental ability of the oocytes is low in pigs, suggesting that a more optimal activation protocol is needed. During fertilization, a sperm-derived factor induces repetitive increases in intracellular calcium, known as calcium oscillations. It is now acknowledged that phospholipase C zeta (PLCζ) has an essential role in inducing calcium oscillations, not only in mammals, but also in several other vertebrates. Therefore, if PLCζ is used as a stimulus for oocyte activation, the efficiency of oocyte activation can be improved. Recently, we found that equine PLCζ (ePLCζ) has higher activity than those of other mammalian species to be studied. In the present study, we examined whether injection of ePLCζ complementary RNA (cRNA) improves the activation of round spermatid-injected oocytes in pigs. First, we examined whether ePLCζ is expressed in round spermatids. Porcine round spermatids were isolated from adult testes, and immunostaining using anti-PLCζ antibody was carried out. The PLCζ was localised at the head and tail in mature sperm, and a part of the round spermatid was also stained. Next, we evaluated the developmental ability of round spermatid-injected oocytes activated by different protocols (electrical pulses v. injection of ePLCζ cRNA). The cytoplasts were then injected with round spermatids. One hour later, the oocytes were divided into two groups. In group 1, the oocytes were activated by a direct current pulse (150 V mm–1 and 60 µs). In group 2, the oocytes were injected with ePLCζ cRNA as follows: the reagent (0.1 µg µL–1) was diluted in injection buffer [100 mM KCl and 10 mM HEPES (pH = 7.0)], loaded into glass micropipettes by aspiration, and delivered to the ooplasm by pneumatic pressure (Narishige, Tokyo, Japan). Each oocyte received 3 to 10 pL (1 to 3% of the total volume of the oocyte). After the stimulations, oocytes were cultured in PZM-5 under 38.5°C in a humidified incubator (95% air, 5% CO2). In the ePLCζ-injected group, rates of pronuclear formation (n = 22/32, 68.8%) and blastocysts (n = 2/43, 4.7%) were higher than those in the electrical pulse-treated group (n = 9/41, 22%; and n = 0/51, 0%, respectively; P < 0.05). In conclusion, our data suggest that injection of PLCζ is effective for activation of round spermatid-injected oocytes in pigs.

Safety Pharmacology Screening: Practical Problems in Drug Development

1997 ◽  
Vol 16 (1) ◽  
pp. 41-65 ◽  
Author(s):  
Lawrence I. Mortin ◽  
Christopher J. Horvath ◽  
Michael S. Wyand
Keyword(s):  
Nonhuman Primates ◽  
Animal Species ◽  
Smooth Muscles ◽  
Electrolyte Balance ◽  
Large Animal ◽  
Toxicity Studies ◽  
General Pharmacology ◽  
Pharmacologic Effect ◽  
Immunologic Diseases ◽  
Species Specific

Undesired pharmacologic activities of novel drugs or biologies may limit development of a therapeutic prior to the characterization of any toxicologic effects. In rodent species, general pharmacology assays have traditionally been used to screen new agents for pharmacologic effects on the central and peripheral nervous systems, the autonomic nervous system and smooth muscles, the respiratory and cardiovascular systems, the digestive system, and the physiologic mechanisms of water and electrolyte balance. In large animal species, such as dogs and nonhuman primates, smaller numbers of animals per study limit their use for screening assays, but these species may play an important role in more detailed mechanistic studies. For drugs and biologies that must be tested in nonhuman primates because of species-specific action of the test agent, functional pharmacologic data are often collected during acute or subacute toxicity studies. This requires careful experimental design to minimize any impact pharmacologic effects or instrumentation may have on the assessment of toxicity. In addition, with many new therapies targeted at immunologic diseases, the pharmacologic effect of therapeutics on the immune system presents new challenges for pharmacologic profiling. The application of pharmacology assays by organ system in both rodent and large animal species are discussed, as well as practical issues in assessing pharmacology endpoints in the context of toxicity studies.

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.

scholarly journals Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 9-14 ◽  
Author(s):  
H. Watanabe ◽  
H. Tateno ◽  
H. Kusakabe ◽  
T. Matsuoka ◽  
Y. Kamiguchi ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chromosome Aberrations ◽  
Chromosome Complement ◽  
Chromosome Analysis ◽  
Oocyte Activation ◽  
Mouse Oocytes ◽  
Structural Chromosome ◽  
Sperm Nuclei ◽  
Immotile Spermatozoa

SUMMARYPrior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3–97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2–93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.

scholarly journals Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes

2012 ◽  
Vol 27 (6) ◽  
pp. 1768-1780 ◽  
Author(s):  
S.-Y. Yoon ◽  
J. H. Eum ◽  
J. E. Lee ◽  
H. C. Lee ◽  
Y. S. Kim ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Phospholipase C ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Human Oocytes

scholarly journals The exit of mouse oocytes from meiotic M-phase requires an intact spindle during intracellular calcium release

1995 ◽  
Vol 108 (1) ◽  
pp. 143-151 ◽  
Author(s):  
N.J. Winston ◽  
O. McGuinness ◽  
M.H. Johnson ◽  
B. Maro
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Calcium Transient ◽  
Oocyte Activation ◽  
High Concentration ◽  
Microtubule Inhibitor ◽  
Mouse Oocytes ◽  
M Phase ◽  
Single Spike ◽  
Calcium Spiking

To study the role of the metaphase spindle during the period of oocyte activation, mouse oocytes were fertilised or activated parthenogenetically in the presence or absence of the microtubule inhibitor nocodazole. In both cases, nocodazole caused the disappearance of the spindle and prevented the passage of the oocytes into interphase. However, the calcium spiking responses of the oocytes were not affected by nocodazole, being repetitive after fertilisation and a single spike after activation. If, after their activation or fertilisation in nocodazole, oocytes were later removed from the drug, only those that had been fertilised progressed into interphase. This progress was associated with continuing calcium spiking. Moreover, both the spiking and the progress to interphase could be blocked or reduced in incidence by removal of external calcium or addition of 5,5′-dimethyl BAPTA-AM. Oocytes that had been activated by ethanol in the presence of nocodazole and then removed from it, to allow re-formation of the spindle, only progressed into interphase if given a second exposure to ethanol, thereby eliciting a second calcium transient. These results show that exit from meiotic M-phase requires the simultaneous presence of a fully intact spindle during the release of calcium and that those factors leading to the degradation of cyclin B are only activated transiently. Since cyclin is being degraded continuously in the metaphase-II-arrested mouse oocyte and since this degradation is microtubule-dependent, these data suggest that the superimposition of a high concentration of intracellular calcium is required to tilt the equilibrium further in favour of cyclin degradation if exit from M-phase is to occur.

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.

scholarly journals CASA-Mot technology: how results are affected by the frame rate and counting chamber

2018 ◽  
Vol 30 (6) ◽  
pp. 810 ◽  
Author(s):  
Daznia Bompart ◽  
Almudena García-Molina ◽  
Anthony Valverde ◽  
Carina Caldeira ◽  
Jesús Yániz ◽  
...  
Keyword(s):  
Animal Species ◽  
Semen Analysis ◽  
Mammalian Species ◽  
Frame Rate ◽  
Human Semen ◽  
Kinematic Parameters ◽  
Sperm Analysis ◽  
Counting Chamber ◽  
Different Depths ◽  
Species Specific

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10 µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20 µm . The frame rate was previously limited by the hardware, although changes in the number of images collected could lead to significant variations in some kinematic parameters, mainly in curvilinear velocity (VCL). A frame rate of 60 frames s−1 is widely considered to be the minimum necessary for satisfactory results. However, the frame rate is species specific and must be defined in each experimental condition. In conclusion, we show that the optimal combination of frame rate and counting chamber type and depth should be defined for each species and experimental condition in order to obtain reliable results.

scholarly journals Intracytoplasmic Sperm Injection in Cattle

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 198
Author(s):  
Veena Unnikrishnan ◽  
John Kastelic ◽  
Jacob Thundathil
Keyword(s):  
Phospholipase C ◽  
Intracytoplasmic Sperm Injection ◽  
Animal Species ◽  
Reproductive Technology ◽  
Oocyte Activation ◽  
Cattle Breeding ◽  
Future Directions ◽  
Low Efficiency ◽  
Bovine Species ◽  
Matured Oocyte

Intracytoplasmic sperm injection (ICSI) involves the microinjection of sperm into a matured oocyte. Although this reproductive technology is successfully used in humans and many animal species, the efficiency of this procedure is low in the bovine species mainly due to failed oocyte activation following sperm microinjection. This review discusses various reasons for the low efficiency of ICSI in cattle, potential solutions, and future directions for research in this area, emphasizing the contributions of testis-specific isoforms of Na/K-ATPase (ATP1A4) and phospholipase C zeta (PLC ζ). Improving the efficiency of bovine ICSI would benefit the cattle breeding industries by effectively utilizing semen from elite sires at their earliest possible age.

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