Elsevier

Theriogenology

Volume 69, Issue 5, 15 March 2008, Pages 546-555
Theriogenology

Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle

https://doi.org/10.1016/j.theriogenology.2007.11.001Get rights and content

Abstract

This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test was correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes.

The expression of Bcl-2 and Bax genes was estimated on the mRNA and protein levels, respectively using real-time PCR and Western-blotting. To evaluate developmental competence of these oocytes, cumulus–oocyte complexes classified as BCB+ (low activity of G6PDH), BCB− (high activity of G6PDH) and Control were used for in vitro embryo production.

In immature oocytes, the Bax transcript level in BCB− oocytes was significantly higher (P < 0.001) in comparison to Control. In mature oocytes, the Bcl-2 transcript level was significantly lower in BCB+ oocytes (P < 0.01) and in BCB− oocytes (P < 0.05) in comparison to Control. However, no relation was found between the activity of G6PDH and the expression of the Bcl-2 or Bax proteins, both in immature and mature oocytes.

Our results on the transcript level seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. However, results obtained at the protein level did not confirm this conclusion. The usefulness of the BCB test as the indirect marker of apoptosis seems to be questionable. The lack of significant differences in the blastocyst rates developed from BCB+ and Control oocytes decreases the validity of BCB test in IVP technology.

Introduction

There are now several evidences suggesting that the intrinsic quality of the oocyte is the key factor in determining the proportion of oocytes developing to the blastocyst stage [1], [2], [3]. Selection of high quality oocytes is highly desirable for in vitro embryo production (IVP) as well as for procedures requiring extensive manipulations such as pronuclear microinjections, intracytoplasmic sperm injection, and nuclear transplantation in order to ensure a high developmental competence after embryo transfer [4].

Immature oocytes in cattle, as in other mammals, are routinely selected for IVM on the basis of the visual assessment of morphological features such as thickness and compactness of the cumulus investment and the homogeneity of the cytoplasm. Although morphological criteria provide reasonable means of identifying oocyte quality and suitability for IVM, there is considerable variability among oocytes and morphological criteria are not sufficient for the identification of oocytes appropriate for in vitro development to the blastocyst stage. Such conclusions can be drawn based on results showing the frequent failure of up to 60% of IVM/IVF oocytes trying to reach the blastocyst stage in vitro, both in cattle and in other mammalian species [1], [5], [6], [7], [8].

It has been demonstrated that brilliant cresyl blue (BCB) can be used for the selection of competent oocytes of pigs, goats and cattle [1], [9], [10], [11], [12], [13], [14]. The test allows one to determine the activity of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme synthesized in growing oocytes but inactive in the oocytes that have finished their growth phase. This enzyme is a component of the pentose phosphate cycle which provides energy for nucleotide synthesis and provides much of the NADPH, serving as a hydrogen or electron donor in biosynthetic reactions such as fatty acids formation [15]. Brilliant cresyl blue is a dye that can be broken down by G6PDH [15]. Oocytes undergoing growth and those that have completed their growth phase will therefore have different levels of G6PDH. Oocytes lacking G6PDH reduce the dye stain blue.

The early developmental program embedded in the oocyte through the accumulation of RNA and proteins is likely to be responsible for the proper execution of the embryonic genome activation and further development. However, the developmental potential of an oocyte may be affected by several factors. One of them may be the apoptotic process, as follicular atresia occurs at all stages of development during life-span. It has been earlier observed that the developmental potential of BCB+ oocytes was higher than that of BCB− and Control oocytes [1], [10], [11], [12], [14]. Considering these observations we hypothesized that BCB+ oocytes might show higher expression of anti-apoptotic genes in comparison with Control and BCB− oocytes. If this hypothesis turned out to be true, the BCB test could also be used as an indirect marker of apoptosis in immature oocytes. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes.

There are two major pathways of apoptosis: the death receptor pathway and the mitochondrial pathway (reviewed by [16]). The mitochondrial pathway is regulated by the pro- and anti-apoptotic members of the Bcl-2 family of proteins and leads to the cytosolic realization of mitochondrial intermembrane space proteins that can trigger caspase-dependent death pathways [17], [18]. The role of Bcl-2 family members in apoptosis in female gonads has been widely reviewed by Kim and Tilly [19]. It has been suggested that the ratio of pro-apoptotic gene Bax and the anti-apoptotic gene Bcl-2 can determine whether a cell will be protected from apoptosis or die from it [20].

The expression of Bcl-2 and Bax proteins in oocytes along with embryos of different quality and stages has been assessed by western-blotting analysis by Yang and Rajamahendran [21]. They observed the high expression of Bcl-2 in good quality oocytes and embryos, while it was hardly detectable in denuded oocytes. On the contrary, the expression of Bax was found in all types of oocytes and the highest level was shown in denuded oocytes. Therefore, Yang and Rajamahendran [21] concluded that the ratio of Bcl-2 to Bax may be used to predict the tendency of oocytes towards either survival or apoptosis. In many studies estimating the expression of apoptotic genes as a quality marker for oocytes and embryos, assessment of the expression of apoptotic genes is more frequently performed at the RNA level than at the protein level [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32].

This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test is correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. The combined morphological and biochemical selection may enable the improvement of the IVP efficiency.

Additionally, to assess the effect of BCB on oocyte developmental competence, the COCs subjected to BCB staining and Control were used for IVP.

Section snippets

Materials and methods

Unless otherwise indicated, all plastic ware i.e., culture vessels, dishes and tubes used in our experiments were obtained from Nunc, Wiesbaden, Germany and all chemicals and media were purchased from Sigma, Poznań, Poland.

Activity of G6PDH in immature bovine oocytes

A total number of 4671 immature oocytes were subjected to BCB test (in the Experiments 1 and 2). In the pool of COCs subjected to BCB staining, the mean rates of the oocytes with low (BCB+) and high (BCB−) activity of G6PDH was 70% and 30%, respectively (Table 2).

The interaction among activity of G6PDH in oocytes and their developmental competence

As it is presented in Table 3, significant differences were shown in embryo cleavage rates among the BCB+ and BCB− oocytes (P < 0.001), the BCB+ and Control oocytes (P < 0.05), and the BCB− and Control oocytes (P < 0.05). A significantly

Discussion

As it was demonstrated in Experiment 1, the developmental competence following IVM/IVF of oocytes with a low activity of G6PDH (BCB+) and Control was significantly higher in comparison to oocytes showing a high activity of G6PDH (BCB−). These results are in agreement with similar experiments in cattle [1], [11], [12]. However, in the experiments mentioned above, the blastocyst rates of BCB+ oocytes were significantly higher in comparison to Control. That differences were not observed in our

Acknowledgements

This research was supported by the statutory activity of the National Research Institute of Animal Production, project no. 3322.1; by the State Committee for Scientific Research as a Doctoral Dissertation Grant 2 PO6D 007 28; and by the Scientific Net “Animal Reproduction Biotechnology”.

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