Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence

Abstract

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus–oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus–oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus–oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus–oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus–oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.

Introduction

Despite the progress made in optimizing culture systems for bovine oocyte in vitro maturation, fertilization and embryo production, only about 40% of oocytes retrieved from abattoir ovaries may develop to the transferable stage [1], which could partially be due to the use of oocytes of inferior quality [1], [2], [3], [4]. However, the underlying causes of oocyte inferiority are poorly understood. One possibility is that oocytes of inferior quality are predestined to undergo apoptosis. Apoptosis, or programmed cell death, is cell self-destruction under physiological control [5]. Typical features of apoptotic cells are cell shrinkage, plasma membrane blebbing, nuclear chromatin condensation and fragmentation, DNA fragmentation and segmentation of the cell into apoptotic bodies [6]. Many proteins, for instance members of the Bcl-2 and caspase family, reportedly participate in the induction, execution or suppression of apoptosis, although the exact mechanisms involved are far beyond our knowledge. About a decade ago, the occurrence of apoptosis in mouse MII stage oocytes was independently reported by Takase et al. [7] and Fujino et al. [8]. This finding was confirmed later on by Perez et al. [9], who successfully detected evidence of apoptosis (DNA fragmentation and caspase activity) in ovulated mouse oocytes. However, in 1998, Van Blerkom and Davis [10] questioned the presence of apoptosis in mouse and human oocytes since DNA strand breaks were rarely found in newly ovulated and cultured oocytes of both species. They subsequently stated it was premature to conclude that apoptosis occurs in ovulated oocytes, which challenges the results of previous researchers and leaves the existence of oocyte apoptosis unresolved.

Cumulus cells play a critical role in oocyte maturation and fertilization by releasing and mediating signals to oocytes [11]. Whether the occurrence of apoptosis in the cumulus–oocyte complex (COC) has an impact on oocyte development potential is unclear, since contradictory reports in various species fail to clarify if apoptosis really takes place in COC. For instance, studies of Lee et al. [12], Mikkelsen et al. [13] and Zeuner et al. [14] showed that human and bovine cumulus cells were subjected to apoptosis, while others demonstrated the absence of apoptosis in cumulus cells of rats [15], pigs [16], and cattle [17], [18]. Among these authors, only Lee et al. [12] investigated the relationship between apoptosis in cumulus cells and the outcome of human embryo in vitro culture, and concluded that the incidence of cumulus cells apoptosis can be used in predicting oocyte quality, outcome of in vitro fertilization-embryo transfer (IVF-ET), and age-related decline in fertility. However, it is unknown if such a correlation is present in cattle as well.

In order to clarify these discrepant findings of oocyte and cumulus apoptosis, it was the aim of this study to investigate [1] whether typical apoptosis features can be found in bovine oocytes and their cumulus cells by light microscopy, TUNEL assay and caspase staining; [2] if so, whether there is a relationship between oocyte or cumulus apoptosis and oocyte developmental competence. The terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay is a widely used approach to label and recognize DNA fragmentation by using the terminal deoxyribonucleotidyl transferase [19], while caspase staining is based on carboxyfluorescein-labeled fluoromethyl keton (KMK)-peptide inhibitor of caspases, which is a general inhibitor of caspase 1–9 and can permeate through the cell membrane and binds covalently to the active caspases [20].