In-vitro maturation technique or IVM plays an important role in animal biotechnology, as this technique has been established as it is recognized for its crucial role in some important aspects such as the clinical applications in human beings, and in expanding some agriculturally important populations and some endangered species. IVM is greatly helpful in the production of embryos for research, productivity in animals, and infertility in human beings. The lack of in-vitro production systems in the pig resulted in the low yields of the blastocysts which in turn lead to a few viable pregnancies. It is important to understand the molecular mechanisms of oocyte maturation to find solutions to the problems such as the poor cytoplasmic maturation characterized by higher rates of polyspermy, and low pronuclear formation.
The molecular mechanisms also help in searching for a sufficient mature oocyte that can be fertilized in-vitro, for the suitable embryos to transfer, for a gene-pool bank of frozen embryos, and for the application of these techniques in genetic engineering, The period of oocyte maturation is lengthy in pigs, that makes it suitable domestic model for the IVM biochemical studies of the maturation of the oocyte. For the investigation and the analysis of the IVM method, it is important to have knowledge of the regulatory mechanisms that play a key role in the maturation of the oocytes of the pig. Protein kinase C (PKC) is an important signaling molecule that influences mammalian reproduction and meiotic maturation. As per the evidence from different laboratories, PKC influences the meiotic maturation of the oocytes of the pig.(Su et.al,1999). It is important to take up a study that improves the basic knowledge on the IVM process and the role of PKC in meiotic maturation. The objective of the present study is to investigate the importance of the expression of protein kinase C, the effect of the inhibitors of the isoforms of the PKC, and the key role of the in-vitro maturation of the oocytes in the animal industry. The investigations would help to enhance animal production with the above procedures and to extend the studies in human beings if they are safe and effective.
Objectives of the study
- Investigate the expression of the isoforms of the PKC in the invitro-matured oocytes of the pig at different stages of meiotic maturation by using the western blotting technique.
- To investigate the role of calcium-dependent and calcium-independent protein kinase C on the parthenogenetic activation of pig oocytes.
Goals of research
- The first goal of my research was to show the expression of the seven chosen PKC isoforms from the conventional and novel PKC’s in pig oocytes at three predefined states of growth namely GV.MI, and MII, through the immunochemical reactions.
- My second goal was to examine the specific role of the individual PKC isoforms during the parthenogenic activation of the mature oocytes of the pig.
Materials and Methods
We conducted several experiments to assess the in-vitro maturation capacity of the pig oocytes and the effect of the inhibitors of the PKC on the activation of oocytes during the period of meiotic maturation. There were three replications for each experiment.
The intention of this study is to evaluate a procedure for the in-vitro maturation of the pig oocytes at three stages of IVM i.e GV, MI, and MII, and the expression of the isoforms of PKC during these stages of the IVM.
The second study is to detect the effect of the PKC inhibitors on the activation of the oocytes during the stages of IVM.
Collection of the oocytes
Pig ovaries of unknown reproductive history were collected from the local slaughterhouse in a thermos flask that contains a physiological solution at 39oc. cumulus-oocyte complexes (COC) were aspirated from antral follicles(2-5mm in diameter) with a 20 gauge needle that is fixed to a 20ml disposable syringe. The follicular fluid that is collected is pooled in Petri dishes. The COC is selected in the presence of a multilayered compact cumulus investment and homogenous cytoplasm in the IVM medium.
Maturation of the oocytes
The cumulus-oocyte complexes are washed three times in a maturation medium. The maturation medium is a tissue culture medium M-199 ( Gibco BRL, Life Technologies, Paisley, Scotland). In each of the Petri dishes containing the above medium is added 50 oocytes in 3ml of in-vitro maturation medium, cultured at 38oC in a humified atmosphere of 5% CO2 in the air and kept for 0hr, 24h, and 48hr for the GV, MI, and MII stages of maturation respectively.
Different doses of the inhibitors of the PKC were added to the maturation medium, and bisindolylameimide I(0,1,2,5,10,20,nM) rottelerin, (0,1,2,5,10,20μM), G-66976,(0,1,2.5,5,10,25 μM) and Hispidin(0,2,20,200,2000 μM) dissolved in dimethyl sulfoxide are used as inhibitors. These are added to the culture medium and kept for 48hrs along with the control without the PKC inhibitors.
Activation of the oocytes
After the 48hr period of the cultivation of oocytes, the oocytes free of cumulus cells are placed in the medium with respective inhibitors to ensure whether there is any inhibition.
Samples for the analysis by western blotting technique for the two experiments were prepared. Oocytes from the stages GV, MI, MII were denuded from the cumulus cells and washed with TBS( Tris Hcl of PH7.5-10, 150mM NaCl), and lysed in 50μl lysis buffer (10% Triton X, 10% SDS, PBS supplemented with the protease inhibitor cocktail. It is centrifuged three times that is washed with acetone each time. The sample is then kept at a deep freeze temperature of -20oC.
The chromosomes of GV, MI, MII stages are evaluated on the oocytes after 0h, 24h, and 48h for the incubation on the maturation medium. Oocytes denuded off the cumulus cells are washed with the PBS solution and mounted on the glass slide with the coverslip that is suspended with Vaseline, and the slide is fixed with ethanol-acetic acid(3:1), for 48 hours. After the fixation process, the oocytes are stained with 1% aceto-arcein and examined with Hoffmann modulation contrast equipment. Basing on the duration of incubation, the counted oocytes are expressed as a percentage. The percentage of GV(with clear nuclear membranes) and the GVBD(without nuclear structures) per total oocytes were cultured and calculated.
SDS-PAGE and Western Blot analysis
The total protein was extracted from the oocytes was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (SDS-PAGE), with 4% stacking gel and 10% separating gel. A reminiscent fluorescent light was caught on RTG film that is sensitive to blue light. PKCα, βI, βII, γ, δ are included in the experiment as positive controls. Molecule masses are controlled based on the marker of the molecular mass. The results of the experiment are evaluated visually.
Detection of the PKC isoforms
This experiment is done to examine the ability of the nuclear maturation, cumulus expansion, and the expression of the PKC isoforms during the invitro meiotic maturation on the M-199 medium, to evaluate the progression of the meiotic maturation of the oocytes, and the expression of the PKC isoforms. The pure protein isotypes of the PKC were used as controls. Pure proteins were used in the experiments with PKCα, βI, βII, γ, δ. For the isoforms ε, and η pig leukocytes are used as a positive control.
Influence of the PKC inhibitors on the activation of the oocytes of the pig:
The influence of the PKC isoform inhibitors on the activation of the oocytes by ionophore A23187 was investigated. The oocytes were treated with calcium ionophores when they have been exposed to the effects of inhibitors for 30 minutes prior to activation and after activation for 23.5 hrs in the presence of the PKC inhibitors.
After conducting four replicas of the experiment, that focus on the influence of the PKC inhibitors on the oocytes of pigs, the results were compiled and evaluated by the χ2 analysis.(Li et.al,2004). The difference between the percentage of the oocytes that reached the end of the nuclear maturation in all the replicated experiments and the average of the compiled data was more than 2.5%. The level of the statistically significant difference (P) was less than 0.05.
The first part of our study comprises monitoring the expressions of the protein kinase C isotypes during the in vitro meiotic maturation of the pig oocytes with the western blot analysis technique. The isoforms of the PKC used for our study were four conventional PKCs i.e α, βI, βII, and γ and the three novel types of isoforms of PKC used for the study include δ, ε, η respectively.
Expression of the PKC isoforms in the mature oocytes of pig
The IVM stages of GV, MI, and MII were observed with 96% of the cultured oocytes at appropriate periods of the culture. Of the four conventional isoforms PKC, no isoform was observed in three stages of maturation of the cultured oocytes. Of the three novel isoforms of the PKC, a small amount of PKC δ protein was observed with a molecular mass close to 80kDa which corresponds with the typical molecular mass of PKC δ protein (77.5kDa).
Role of PKC isoforms in the activation of oocytes
In this experiment, we observed the influence of PKC isoforms in inhibiting the activation of the oocytes. When the PKC isoforms are treated with the inhibitor, bisindolylameimide I, there was a suppression of activity. The suppression of the activity of the oocytes was significant with the increasing concentrations of the bisindolylameimide. Oocytes, when treated with the ionophore A23187, and cultured along with the inhibitor Gδ6976 and histidine for the isoforms of PKC namely PKCα, and PKC βI there was no suppression of activity. Rottlerin, an inhibitor for the isoform of PKCδ, suppressed the activation of oocytes even though it is treated with the ionophore of calcium.
There was a reduction in the number of the activated oocytes and it is significant, and there was an inverse proportionality to the concentration of the Rottlerin used. 96% of the cultivated oocytes. In a control experiment, the role of mature oocytes up to the MII stage, the role of the oocytes that are activated spontaneously, and the number of the activated oocytes after treatment with the calcium oocytes are verified. It is observed that after 48hrs, 96% of the cultured oocytes completed the maturation till the MII stage. Maturation of the cultured oocytes was not observed after the removal of the cumulus cells and subsequent cultivation for 24 hrs. If the cultured oocytes devoid of cumulus cells are cultured further for 48 hrs, and exposed for 10 minutes to a medium with fetal bovine serum and kept for 24 hrs and there was no activation of the oocytes. The oocytes devoid of cumulus cells, if exposed for 10 minutes to the effects of 50μM of calcium ionophore A23187, and cultured for 24 hrs in vitro, activation of 72% of the oocytes was observed.
Pig oocytes, during the growth period, have gone through a series of morphological and ultrastructural changes that are necessary for them to acquire the ability for the synthesis of necessary proteins so that the cells acquire meiotic competence for the complete meiotic process.
Expression of the PKC isoforms in pig oocytes
In our studies, we observed the expression of only the novel types of PKC isoforms, and we did not observe any of the conventional PKC isoforms. Some of the studies by researchers have reported the presence of the conventional isoforms in the fully matured oocytes. (Gangeswaran and Jones,1997, Raz.et.al,1998, Luria et.al, 2000, Downs et.al, 2001, Viveiros et.al,2001, Fan et.al, 2002). In our studies, we observed that PKCδ plays an important role in the activation of the oocytes. The difference between the results of Fan et. al,2002, (who detected the conventional PKC isoforms in the maturation of oocytes) and our studies is due to the utilization of different types of chemicals from different producers and a different breed of pig.
The PKC isoform PKCε has not been found so far in the studies by other researchers. These PKC’s are found only in traces with no PKCη in the leukocytes of pig and Gangeswaran and Jones,1997 reported on their studies in a similar manner. The majority of the PKC’s that are identified were in a split form. with a molecular mass close to 50kDa. Members of the PKC family are single-stranded polypeptides with regulatory and catalytic domains.(Coussens et.al). The ability of the Calpain enzymes to split PKC, generating a catalytic subunit that is independent of co-factors indicates an apparent role during the regulation of PKC.(Cressman et.al,2000). On the basis of our studies, we infer that PKCδ may be involved in the suspension of meiosis and regulating the release of the meiotic block before the maturity stage that is MII. The novel types of PKC isoforms were not found in the stipulated forms of pig oocytes.
Influence of PKC inhibition on the activation of pig oocytes
Bisindolylameimide I, an inhibitor to all types of isoforms of PKC, which are calcium-dependent and calcium-independent, inhibits the activation induced by the calcium ionophore A23187. In our studies we observed that there was no blockade of the maturation of the oocytes that did not occur with Hispiridine and Gδ6976, but Rottlerin inhibited the activation. From the above results, we inferred that the PKCδ plays an important role in the activation of the oocytes of the pig. PKCδ independent influence of Rottlerin has been observed in astrocytes. (Susarla and Robinson,2003). Following the activation of oocytes the concentration of Ca+ increases in two ways. (Homa et. al.2003). Fan et.al, 2002 described the translocation of the PKC in oocyte cytoplasm following fertilization and parthenogenetic stimulation by the calcium ionophore A23187. We can support our conclusion with the reports on Wang et.al that the ionophore increases the intracellular levels of calcium ions. On the basis of our studies, we conclude that PKCδ isoform takes part in the activation of pig oocytes.
From our studies, we hypothesized that a number of PKC isoforms are already present in the pig oocytes during their growth and play an important role in the development of oocytes. In our analysis through the western blot technique, we found that the isoform of the PKC was predominantly in a divided form than in an undivided form. In in-vitro maturation, any form of isoforms of PKC namely the conventional or novel was detected in the western blot analysis. Bisindolylameimide I suppressed the activity of the oocytes, and Hispiridine and Gδ6976 were unable to suppress the activity of the oocytes. Rottlerin inhibited the activity of oocytes, and from this, we concluded that calcium-independent PKC’s play an important role in the activation of oocytes.
In our experiment, there was some inconsistency of the results when compared with the results of Fan et. al,2002, so we recommend that using these conditions it is necessary to sort out the differences from the previous studies. I suggest a deep study of the isoforms of PKC in the oocyte maturation as we detected only one type of the isoform of PKC namely PKCδ. The role of PKCδ in oocyte maturation needs further investigation for confirmation. The specific mechanism by which the PKCδ plays an active requires further investigation.
Invitro maturation of the oocytes or IVM is an important technique in animal biotechnology and it has potential uses in the clinical applications of human beings, in the expansion of populations that are agriculturally important and endangered species. The IVM technique plays an important role in the production of embryos. It is necessary to understand the molecular mechanism of oocyte maturation as these techniques are of great help in genetic engineering. The lengthy oocyte maturation period in the pig is suitable as a domestic model for biochemical studies of the maturation of oocytes. It is important to understand that some regulatory mechanisms play a key role in the maturation of oocytes.
As evidenced by different laboratories, meiotic maturation in pigs is influenced by a signaling molecule, PKC, or Protein kinase C. It is important to improve the basic study on IVM and the role that the PKC plays in the meiotic maturation of the oocytes. It is necessary to investigate the expression of different isoforms of PKC (conventional and novel) at different stages of the maturation of the oocytes namely GV, MI, and MII. It is also necessary to study the specific role of the individual isoforms of the PKC’s during the parthenogenetic activation of the maturation of oocytes. There was no expression of the PKC isoforms whether conventional or novel, except a small amount of the novel isoform of PKC namely PKC δ. Inhibitors such as Bisindolylameimide I, which may be calcium-dependent or independent inhibit the activation induced by the calcium ionophore A23187 and there was no inhibition with inhibitors such as Hispiridine and Gδ6976, while Rottlerin inhibits activation. PKC’s which are calcium-independent play an important role in the process of the activation of oocytes.
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