Elsevier

Theriogenology

Volume 57, Issue 5, 15 March 2002, Pages 1523-1532
Theriogenology

Effect of follicular size on meiotic and developmental competence of porcine oocytes

https://doi.org/10.1016/S0093-691X(02)00655-6Get rights and content

Abstract

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three follicular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n=330), medium (3–5 mm, n=373), or large (>5 mm, n=238). The cumulus–oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 μM cysteamine; in vitro fertilized for 18 h in mTBM with 105 frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P<0.05). More oocytes from medium and large follicles were penetrated by spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P<0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P<0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.

Introduction

In vitro production (IVP) of embryos is of interest in different mammalian species, from zootechnical applications and development of new biotechnologies to basic research in physiology and cell biology. This technique includes usually three major steps: in vitro maturation (IVM) of follicular oocytes, in vitro fertilization (IVF) of the matured oocytes with fresh or frozen-thawed semen, and in vitro development (IVD) of the resulting zygotes to the morula or blastocyst stage, which allows their subsequent transfer to the uterus of a synchronized female. After the birth of the first calf obtained from an IVP embryo in 1982 [1], this technique has been proven to be promising to a wide range of large-scale applications. However, in spite of a large quantity of work leading to major improvement of the three steps, the rate of oocytes finally reaching the blastocyst stage still plateaus at around 20–40%, depending on the species [2].

In ruminants, IVP conditions may be very efficient when used with selected oocytes (cattle: [3]; goat: [4]; sheep: [5]). In these species, it is possible to obtain high rates of development of embryos to the blastocyst stage with conventional IVP techniques by using oocytes collected from large follicles or ovulated (in vivo matured) oocytes [6], [7]. This indicates that some important events take place in the oocyte during late follicular growth, leading to its full developmental competence. Oocytes first become able to resume meiosis in early antral follicles, then become able to complete meiosis up to metaphase I and proceed to metaphase II in growing antral follicles at a given diameter, according to the species. First demonstrated in mice [8], this stepwise acquisition of meiotic competence during follicular growth has been confirmed later in ruminants [9]. Beyond meiotic competence, the oocyte has to complete its differentiation by acquiring the ability to support the cytoplasmic maturation and finally the ability to be successfully fertilized and develop into a viable embryo. This terminal differentiation of meiotically competent oocytes occurs at the end of folliculogenesis and is made possible by the meiotic inhibition addressed by the follicular cells to the oocyte. This late period of differentiation has been referred to as oocyte capacitation [10]. In the porcine, Motlik et al. [11], [12] have shown that meiotic competence is also acquired progressively by the oocyte during follicular growth. However, there is no indication of the evolution of developmental competence. Oocytes used for pig IVP are usually collected from a wide range of follicular diameter (3–6 mm) and selected according to the morphology of the cumulus–oocyte complexes (COCs). The heterogeneity of these COCs may partly explain the poor fertilization and developmental results usually observed in pig IVP [2], [13].

The molecular events accounting for the acquisition of developmental competence by the oocyte are not known. RNA transcription and protein synthesis activities are reduced in oocytes of growing antral follicles, however, the oocyte is still producing low quantities of RNA and proteins up to the LH signal of meiotic resumption [14]. These relatively low quantities may be functionally important and account for increasing developmental potential during late follicular growth. If such functionally important factors exist, their knowledge may be a help towards the improvement of in vitro maturation of the oocytes.

The aim of this study was to follow the evolution of oocyte meiotic and developmental competence as well as protein synthesis activity during the growth of antral follicles in the pig. Thus, we compared the maturation, fertilization, and development rates of oocytes collected from follicles of three different size classes and analyzed the protein neosynthesis patterns of such oocytes before and after in vitro maturation.

Section snippets

Materials and methods

Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. The details of methods for in vitro maturation (IVM), fertilization (IVF), and embryo development (IVD) were previously described [15].

Oocyte collection

A total of 941 follicles were dissected during six replicates. Among these follicles, 330 were classified as small (average size of 2.38±0.48 mm diameter); 373 as medium (average size of 3.92±0.48 mm diameter); and 238 as large (average size of 5.43±0.55 mm diameter). The population of large follicles was less represented in these peripubertal gilt ovaries. The average rate of COC recovery was 92%. COC were randomly allocated to the study of maturation, protein neosynthesis, fertilization, or

Discussion

In this study, we have shown that the developmental competence of porcine oocytes increases in parallel with follicular size. In follicles of less than 3 mm in diameter, 44% of the oocytes are already competent to resume meiosis and progress to metaphase II. However, very few oocytes from these small follicles are able to develop to the blastocyst stage after fertilization (3%). This discrepancy between meiotic and developmental competence may explain partly the contrast observed between the

Acknowledgements

A part of this work was supported by EU (Grant # QLK3-CT-1999-00104, Quality of Life). R. Marchal was supported by a grant from Conseil Régional du Centre (#97-298-025). A. Bali-Papp was supported by a grant from OECD (#JA00003425). The authors thank F. Bussière (UEICP, Rouillé, France) for providing frozen boar semen.

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    Present address: University of West Hungary, 9200 Mosnmagyarovar, Hungary.

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