Elsevier

Theriogenology

Volume 67, Issue 4, 1 March 2007, Pages 795-800
Theriogenology

Parthenogenetic activation of domestic cat oocytes using ethanol, calcium ionophore, cycloheximide and a magnetic field

https://doi.org/10.1016/j.theriogenology.2006.10.009Get rights and content

Abstract

The objective of this study was to evaluate parthenogenetic activation of domestic cat oocytes after being exposed to either ethanol, magnetic field, calcium ionophore A23187, or cycloheximide and a combination of these agents. We also wished to evaluate the usefulness of the magnetic field for oocyte activation. In vitro matured oocytes subjected to artificial activation were randomly assigned into eight groups according to activating agents: (1) 10% ethanol; (2) the magnetic field (slow-changing, homogenous magnetic field with low values of induction); (3) 10% ethanol plus magnetic field; (4) 10 μM calcium ionophore A23187; (5) 10 μM calcium ionophore A23187 plus magnetic field; (6) 10% ethanol and 10 μg/mL of cycloheximide; (7) 10% ethanol and 10 μg/mL of cycloheximide plus magnetic field; (8) oocytes were not exposed to any of the activating agents. After activation oocytes were stained with Hoechst 33258 and parthenogenetic activation was defined as oocytes containing pronuclei and second polar bodies or two to four or six nuclei (embryonic cleavage). The total activation rate by using different activation treatments was 40%. The addition of the magnetic field to ethanol or calcium ionophore treatments resulted in increased parthenogenetic activation rates from 47% to 75%, and from 19% to 48%, respectively (P < 0.001). Instead, when the magnetic field was added to ethanol and cycloheximide treatment, activation rate decreased from 48% to 30%. Oocytes activated with magnetic field only gave the lowest activation rate (12%).

We concluded that a magnetic field can be used as an activating agent, and the combination of ethanol and magnetic field is an effective method for domestic cat oocyte activation.

Introduction

In the process of fertilization interaction between a sperm cell and a secondary oocyte triggers off a series of morphological and biochemical transformations, known as oocyte activation. The key mechanism is a calcium signal, shared by most animal species. Several minutes after the penetration of a sperm cell into an oocyte a quick and transitory drawing occurs from intracellular reserve of calcium. This is the calcium collected in endoplasmic reticulum, although extracellular calcium can also be used to supplement the reserve, thus enabling continuation of the calcium signal [1]. The activation leads to meiosis resumption and extrusion of the second polar body. Consequently, pronuclei are formed, DNA synthesis begins and embryonic cleavage is initiated of artificial activation, caused by external stimuli. Parthenogenesis phenomenon does not occur naturally in mammals, although very rarely spontaneous can take place. Artificial activation is a necessary step in the cloning procedure, while somatic cloning can become one of the methods for saving endangered species of animals (including felids).

Although, several publications on artificial activation of feline oocytes exist, most of them are related to activation during cloning procedure. Only few studies had compared different methods of activation and their effectiveness, as measured by the number of activated oocytes and parthenogenetic embryos [2], [3], [4], [5]. The stimulus which triggers oocyte activation, can be a physical, mechanical or chemical agent. Combination of these agents is also applied. One common, universal method has not been developed because the process is highly specific for each species. Cat oocyte activation by electric current or chemical agents (ethanol, calcium ionophore) has been reported, along with the inhibitors of protein synthesis, protein phosphorylation or histone kinases (kinases PF/MAP; cycloheximide, cytochalasine B) [6], [7], [8], [9]. A single electrical stimulation seems to be insufficient to activate domestic cat oocytes, but their exposure to cycloheximide following electrical stimulation improved the efficacy of parthenogenetic development [3].

Domestic cat oocytes used for nuclear transfer have been activated 1–2 h post-fusion by exposing them to 7% ethanol and then culturing in cycloheximide + cytochalasin D. The results in this study demonstrated that 90% of oocytes cleaved to two to six cells and above 13% reached blastocyst stage [10]. In similar study, reconstructed cat oocytes were activated with 7% ethanol (5 min) followed by incubation in cycloheximide for 4 h. Cleavage rates (55–80%) and embryonic development to the blastocyst stage (5–8%) were similar to those observed with the presence of cytochalasin D [8].

Ethanol alone, at concentration 10% has not been used in cats to trigger parthenogenetic activation, while calcium ionophore has been mainly used in combination with other agents.

Cleavage and blastocyst development of in vivo and in vitro matured domestic cat oocytes exposed to two ionophores (A23187 or ionomycin) at two different concentrations (5 or 10 μM) for 3 or 5 min have been evaluated. In that study, higher activation rates were observed when in vivo matured oocytes were used (60% versus 47%). The cleavage on day 2 was higher for oocytes exposed to the ionophore for 5 min rather than for 3 min. Instead cleavage was not affected by the type of ionophore or its concentration [11]. Protein synthesis and protein phosphorylation inhibitors in combination with ionophores have been used for cat oocyte activation. Oocytes were exposed to calcium ionophore A23187 (5 μM) for 5 min, followed by incubation either with cycloheximide (10 μg/mL) plus cytochalasin D (2.5 μg/mL), or with 6-dimethylaminopurine (2.5 mM) for 4–5 h resulting in similar cleavage rates of 88% versus 82%, and blastocyst development 45% versus 28%, respectively [2]. It has been also demonstrated that in vitro matured cat oocytes can be activated after being exposed to ionomycin (5 μM) followed by 5 h incubation in 10 μg/mL cycloheximide. The overall rate of activation for the treated oocytes was 90% [12].

Magnetic field has not been used to induce artificial activation in oocytes of any animal species. The magnetic field can influence movement of calcium ions in the cell. Studies carried out on isolated pituitary cells had demonstrated that calcium concentration in the cells had increased almost twice from the normal concentration after being exposed to the magnetic field at frequency of 50 Hz and induction of 50 μT [13].

Most of the authors point out, however, that the results of experiments are closely connected with the parameters of magnetic fields applied and also depend on the experimental object (cellular model, animals), subjected to magnetic field. The results vary, sometimes very significantly, even for slightly different values of induction, frequency and field form. Biological effects observed may be stronger for weaker fields and inversely, some of them are proportional to the induction value or do not depend on it. Therefore, the choice of parameters is the product of many trials and the final result depends on the field values chosen.

The aim of this study was to activate in vitro matured domestic cat oocytes using ethanol, magnetic field, calcium ionophore A23187, ethanol and cycloheximide and combinations of these agents, as well as to compare their effectiveness and to evaluate magnetic field usefulness for oocyte activation.

Section snippets

Oocyte recovery and maturation

Ovaries from domestic cats were obtained after ovariohysterectomy at local veterinary clinics. Oocytes selected for in vitro maturation were surrounded by compact layers of cumulus cells and dark ooplasm. Ten to fifteen cumulus–oocyte complexes (COCs) were cultured in four-well tissue culture dishes containing 700 μL of TCM-199 supplemented with 0.3% BSA, 1 UI/mL hCG, 0.5 UI/mL eCG (Intervet International B.V., Boxmeer, Holland), 2.2 mM calcium lactate, 0.36 mM sodium pyruvate, 2 mM glutamine, 50 

Parthenogenetic activation by using different activation treatments

A total of 1068 oocytes were activated, and 101 oocytes allocated to the control group. We evaluated one cell activated oocytes and cleaved (two, four and six cell-stage) embryos as the total number of parthenogentic activated oocytes. Table 1 shows the number and percentages of oocytes activated by each treatment.

Parthenogenetic activation by incorporating magnetic field

The efficiency of artificial activation was improved when oocytes were exposed to a combination treatment of magnetic field with ethanol (75%), or magnetic field and calcium ionophore

Discussion

Artificial activation of oocytes is one of the stages of in vitro procedures, which replace activation of an egg cell by sperm in the process of natural reproduction. Numerous comparative studies were carried out in farm animals in respect to different methods of artificial activation, and the most effective ones were selected. Different methods had not been compared for felids and several possibilities have been applied in hitherto studies, but they have not led to find a highly effective

References (23)

  • N.W. Karja et al.

    Effect of cycloheximide on in vitro development of electrically activated feline oocytes

    J Reprod Dev

    (2005)
  • Cited by (20)

    • A comparative study on parthenogenetic activation and embryo production from in vitro matured caprine oocytes

      2013, Small Ruminant Research
      Citation Excerpt :

      Efficient in vitro procedure for oocyte maturation and parthenogenetic development in small ruminants are important for the development of new biotechnologies such as gene-transfer, gene-expression occurring through different modes of parthenogenetic development i.e. haploid or diploid as well as in vitro multiplication of identical embryos. Several methods have been developed for induction of parthenogenetic activation, including calcium ionophore (Funahashi et al., 1994), ethanol (Kharche et al., 2013; Loi et al., 1998), A-23187, calcium ionophore and cycloheximide alone (Nussbaum et al. 1995) or combined with a protein phosphorylation inhibitor, (DMAP) (Liu and Yang, 1999), electrical shock (Kono et al., 1989; Kim et al., 1996), CaCl2 (Machaty et al., 1996), protein kinase inhibitors (Mayes et al., 1995), G protein stimulation (Machaty et al., 1996), cycloheximide (Nussbaum and Prather, 1995) ionomycin (Loi et al., 1998), ultrasound (Sato et al., 2005), ethanol (Loi et al., 1998), strontium (Meo et al., 2004), Ca–EDTA (Zae and Ryoo, 2007) and magnetic field (Max et al., 2007). In traditional method for production of cloned goat embryos, electric pulses were applied for activation of reconstructed oocytes (Shen et al., 2006).

    • Assessment of parthenogenetic embryo production by activation of in vitro matured caprine oocytes with different concentrations of ethanol

      2013, Small Ruminant Research
      Citation Excerpt :

      Artificial activation of oocytes aims to mimic the action of sperm cells during fertilization (Nakada and Mizuno, 1998). Several methods have been developed for induction of parthenogenetic activation, promote an increase in intracellular free calcium concentrations by the release of calcium from cytoplasmic stores, including calcium ionophore (Funahashi et al., 1994), electrical shock (Kim et al., 1996), CaCl2 (Machaty et al., 1996), Ca-EDTA (Lee et al., 2007), protein kinase inhibitors (Mayes et al., 1995), G protein stimulation (Machaty et al., 1996), cycloheximide (CH) (Nussbaum and Prather, 1995), ionomycin (Loi et al., 1998), ultrasound (Sato et al., 2005), ethanol (Loi et al., 1998), strontium (Meo et al., 2004) and magnetic field (Max et al., 2007). These treatments are commonly associated with protein synthesis inhibitors, that prevent cyclin synthesis, such as cycloheximide, and phosphorylation inhibitors, which prevent MPF activation, such as 6-dimethylaminopurine (Susko-Parrish et al., 1994; Loi et al., 1998).

    • Ovarian activity reversibility after the use of deslorelin acetate as a short-term contraceptive in domestic queens

      2012, Theriogenology
      Citation Excerpt :

      Therefore, we inferred that this was due to spontaneous oocyte activation. In that regard, parthenogenesis in cats has been described, with a rate varying from 2 to 35.5% [24,25]. This is apparently the first report of oocyte recovery and viability after short-term contraceptive treatment with deslorelin acetate in domestic cats.

    • Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium

      2010, Theriogenology
      Citation Excerpt :

      Keller et al [25] and Du et al [26] exposed oocytes to ionomycin for 4 min and to A23187 for 5 min, respectively, followed by 4–5 h of cycloheximide treatment, whereas in the present experiment, oocytes were exposed to ionomycin for 8 min and treatment with cycloheximide lasted for 6 h (based on the good results for bovine oocyte activation obtained previously in our laboratory [27]). Grabiec et al [19] obtained 48.43% cat oocyte activation and 18.24% cleavage using a combination of ethanol and cycloheximide. Conversely, the combination of ethanol, cycloheximide, and a magnetic field resulted in 29.68% oocyte activation and only 5.16% cleavage rates.

    View all citing articles on Scopus
    View full text