Effect of the apoptosis rate observed in oocytes and cumulus cells on embryo development in prepubertal goats

Abstract

Oocyte quality is the main factor that determines blastocyst yield; any factor that could affect it, such as apoptosis, could impair subsequent embryonic development. Our aim was to investigate the incidence of apoptosis in prepubertal goat oocytes and cumulus cells, assessed by Annexin-V staining and TUNEL assay, and their effect on embryo development. Oocyte-cumulus complexes (COCs) from slaughtered females were collected and classified depending on COC morphology as: Healthy (H) and Early Atretic (EA). Each one of these groups was classified depending on oocyte diameter: A: 110–125 μm, B: 125–135 μm and C: >135 μm. The COCs were IVM for 27 h, IVF with fresh semen and IVC for 8 days after insemination. Apoptosis analyses were performed before and after maturation. Annexin-positive oocytes decreased with diameter in the EA class (immature oocytes: A: 42.6%; B: 30.3%; C: 21%; IVM-oocytes: A: 17.5%; B: 4.8%; C: 0%), while TUNEL assay showed a decrease of apoptosis in the largest oocytes before and after IVM only in Healthy oocytes (immature oocytes: A: 51.5%; B: 43.3%; C: 12.1%; IVM-oocytes: A: 31.7%; B: 12%; C: 0%). Blastocyst rate increased with increasing oocyte diameter, and it was higher in H than in EA oocytes (Healthy; A: 0%; B: 5.3%; C: 14.4%; Early atretic: A: 0.3%; B: 4.1%; C: 5.1%). Oocyte diameter and COC morphology had no effect on the percentage of apoptosis in blastocyst cells. In conclusion, oocyte developmental competence in prepubertal goats is influenced by oocyte diameter and COC morphology.

Introduction

Apoptosis is a process of programmed cell death that regulates biological processes. In the ovary, apoptosis is responsible for follicular atresia, whereby most follicles present at birth are lost during further development. Follicular atresia can occur at any stage of follicular development, but the follicular compartment in which atresia is initiated differs. Thus, in antral follicles, atresia starts with the degeneration of granulosa cells (Irving-Rodgers et al., 2001) and the oocyte is only affected during the last stage of follicular atresia (Driancourt et al., 1991, Yang and Rajamahendran, 2000, Yang and Rajamahendran, 2002). In contrast, in preantral follicles, cell death is often initially observed within the oocyte (Marion et al., 1968, Reynaud and Driancourt, 2000). Besides the compartment initially affected, the molecular mechanism of granulosa cell death also varies according to follicle size (Alonso-Pozos et al., 2003). The process of atresia occurs continuously from birth until the complete depletion of the follicle pool (Hirshfield, 1991, Hsueh et al., 1994, Kaipia and Hsueh, 1997), and over half the follicles present in an ovary at any given moment are at different stages of atresia (Kruip and Dieleman, 1982). This determines that when oocytes from slaughtered animals are recovered by slicing, most of these oocytes will have arisen from atretic follicles and blastocyst production could be consequently impaired.

Several attempts have been made to establish the factors that confer oocytes developmental competence, such as stage of the oestrous cycle (Hagemann et al., 1999), hormonal patterns (Kruip and Dieleman, 1982), composition of follicular fluid (Chang et al., 2002, Chiu et al., 2002, Anifandis et al., 2005, Wunder et al., 2005), follicular diameter (Lonergan et al., 1994, Blondin and Sirard, 1995, Crozet et al., 1995, Hagemann et al., 1999) and follicular atresia (Blondin and Sirard, 1995). However, once the oocytes have been harvested from the ovaries of slaughtered female animals for in vitro embryo production, it is difficult assess these variables to select the most competent oocytes. In addition, when the oocyte donors are prepubertal goats, oocyte recovery involves slicing the ovarian surface, rather than follicular aspiration as used in adults. This method serves to recover a large number of oocytes but regardless of the diameter of the follicles or their extent of atresia. Consequently, the oocyte population recovered from prepubertal goats is very heterogeneous, and thus must be subjected to a strict selection procedure to identify the most competent oocytes.

The close contact between cumulus cells and oocyte through gap junctions allows the bidirectional interchange of molecules (Tanghe et al., 2002). Therefore, any factor that could affect cumulus cells, such as an apoptotic state, might also reflect a lower oocyte quality and, as a consequence, in vitro embryo production may be impaired. In general, it is accepted that cumulus-oocyte complexes (COCs) that come from non-atretic follicles show compact cumulus cell layers and an homogeneous oocyte cytoplasm (de Wit et al., 2000) as well as showing higher developmental competence (Lee et al., 2001, Zeuner et al., 2003, Corn et al., 2005, Yuan et al., 2005). Some studies have, nevertheless, provided conflicting results concluding that COCs showing mild signs of atresia lead to higher blastocyst rates in cows (Hazeleger and Stubbings, 1992, Blondin and Sirard, 1995, de Wit et al., 2000, Boni et al., 2002, Feng et al., 2007).

Apoptosis in oocytes could also be a good marker of oocyte quality and its capacity to develop into a viable embryo. Indeed, there is some support for the idea that apoptosis in the oocyte can also affect embryo quality, because of the presence of molecules that regulate the apoptotic mechanism in the maternal mRNA stored in the oocyte (Jurisicova et al., 1998, Exley et al., 1999, Metcalfe et al., 2004). This mRNA is used to maintain oocyte maturation, fertilization and embryo development until the embryonic genome is activated (Bachvarova, 1992, Gandolfi and Gandolfi, 2001). The maternal genotype, through apoptosis, is also thought to play a significant role in cell fragmentation of the embryo (Han et al., 2005). Apoptosis in the blastocyst is a feature of normal development (Hardy, 1997) although a high incidence of apoptosis in the blastocyst will compromise embryonic development and may lead to abnormalities in the foetus (Brison and Schultz, 1997). Few studies, however, have attempted to identify the characteristics of the oocyte related to apoptosis, and only oocyte diameter has been correlated with the level of atresia in the COC (de Wit and Kruip, 2001).

The present study in the goat was designed to: (1) correlate three oocyte diameters (<125 μm, 125–135 μm and >135 μm, based on the classification by Crozet et al. (1995)) and COC morphology with apoptosis in oocytes and cumulus cells; and (2) examine the relationship between oocyte diameter, COC morphology, cumulus cell apoptosis and embryo development. Apoptotic cells were determined using two methods: the TUNEL assay, to identify cells at the later stages of apoptosis by detecting fragmented DNA through dUTP nick-end labelling (Gavrieli et al., 1992); and Annexin-V staining, to detect early apoptotic cells through loss of membrane asymmetry (revised by van Engeland et al., 1998).