Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes
Introduction
Cumulus cells are involved in growth and maturation of oocytes. In primary follicles which contain growing oocytes, the surrounding granulosa cells start to proliferate and can be distinguished as two specific populations during antrum formation: (i) cumulus granulosa cells, which enclose the oocyte with the corona cells as the innermost layers; and (ii) mural granulosa cells which line the follicular wall (Buccione et al., 1990). These cumulus and mural granulosa cells then, together with the oocyte, form a gap-junction-mediated syncytium, which is lined between the oocytes and corona cells. This syncytium also exists between the granulosa cells, to form a communication pathway between the cells, and is essential for oocyte growth to proceed. The granulosa cells then supply the oocytes with nutrients and connect them to the external environment. In fact, the cumulus gap-junctions provide membrane connections which allow for the quick transfer of small metabolites and regulatory molecules into the oocyte. This property of cumulus cells, together with their specific metabolizing capacity, is of major importance in the regulation of oocyte maturation (Redmer and Reynolds, 1996).
The cumulus cells of oocytes play important roles in e.g. the in vitro maturation and oocyte competence (Mori et al., 2000). In cattle, when cumulus cells were removed before maturation, a significant reduction was recorded in the oocyte maturation, fertilization and in vitro developmental rate (Zhang et al., 1995). An extremely heterogeneous population of the cumulus–oocyte complex (COC) is generally collected from ovaries, to be used for in vitro procedures. Sources of variation in the in vitro embryo production (IVEP) may be the age of these cells, the growth stage, and the stage of atresia of the corresponding follicle population (Boni et al., 2002).
The relationship between the ovarian follicle population and meiotic competence of bovine oocytes have been studied by considering the effect of parameters such as the stage of the estrous cycle (Tan and Lu, 1990), hormonal patterns (Kruip and Dieleman, 1982) and the degree of atresia of the follicle and ovarian morphology (Gandolfi et al., 1997). An analysis of these parameters has thus provided general information, without solving the key question of what the oocyte needs to acquire meiotic competence – and whether follicular activity can be manipulated to improve IVEP efficiency (Boni et al., 2002).
The COC morphology is generally related to the degree of atresia of the follicle that it contains. Wurth and Kruip (1992) distinguished three COC morphological grades or scores, regarding the cumulus cells surrounding the oocytes. These were designated as follows: grade (I) with ≥5 layers of dark cumulus cells, grade (II) with ≤5 layers of clear cumulus cells and grade (III) with expanded or partial cumulus cells. This gross and simple classification system generally avoids the wasting of time in follicle dissection and evaluation, and it provides consistent information regarding the in vitro development potential of the different COC grades.
One of the most important factors affecting oocyte competence and early embryonic development is the cytoplasm quality. It has been suggested that the cumulus–oocyte-junction networks are responsible for the cytoplasmic maturation. In spite of the numerous data available on the function of the cumulus oophorus during oocyte maturation and ovulation, the role of the cumulus oophorus during mammalian fertilization still needs to be clarified (Tanghe et al., 2002). To current knowledge, there has been no explanation regarding the relationship between COC grades and cumulus expansions or cytoplasmic maturation. This may be due to the increment of nutrients or some regulatory molecules, like growth factors and metabolites, such as pyruvate. The aim of this study was thus to investigate whether the various layers of cumulus cells in the COCs improve the developmental ability of the ovine oocytes in terms of cumulus expansion, meiotic competence and cytoplasmic maturation.
Section snippets
Materials and methods
This study was conducted as a part of the ovine IVF program organized by University of Tehran, Department of Animal Science between the 31st September and the 5th of November (autumn). A total of 418 ovine ovaries were recovered post-mortem at the local abattoir in Karaj city, Iran. Ovaries were harvested at evisceration and immediately placed in a saline solution (0.9% NaCl), with penicillin/streptomycin (50 μL), at 37 °C, and transported to the laboratory within 1–2 h, prior to aspiration. All
Results
The oocyte recovery and IVM rate is set out in Table 1. A total of 535 ovine oocytes were evaluated in the trial. The percentages of COC expansion following 24 h of incubation was 53.0 ± 1.0, 36.3 ± 2.2 and 16.3 ± 1.8% (p < 0.05) for group I to III (group IV being denuded oocytes), respectively (Table 1). The rate of nuclear maturation of the oocytes (% M-II) was 77.0 ± 2.7, 77.2 ± 1.9, 53.0 ± 2.1 and 2.7 ± 1.1% (p < 0.05) for group I to IV, respectively (Table 1). The results of the cytoplasmic evaluation
Discussion
In the present study, the recovery rate of quality I and II oocytes using a flow rate of 10 mL water/min was 96%. Morton et al. (2008) recorded similar results (86% recovery rate) under similar conditions. However, Rodriguez et al. (2006) reported a low recovery rate of approximately 60%, for post mortem ovine oocyte retrieval. Based on the results of Shirazi et al. (2005) the recovery of oocytes considered viable for the IVM procedure was higher following the slicing technique, compared to
Conflict of interest
None of authors of this manuscript have a financial or personal relationship with other people or organizations that could inappropriately influence or be bias to the content of the manuscript.
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