Use of polarized light microscopy in porcine reproductive technologies

Abstract

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) could be useful for a non-invasive evaluation of the meiotic spindle and may allow removal of nuclear structures without fluorochrome staining and ultraviolet exposure. In this study, PLM was used to assess its potential application in porcine reproductive technologies. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein in in vitro-matured porcine oocytes; to examine its effects on the oocyte developmental competence; to select oocytes based on the presence of the meiotic spindle detected by PLM; and to assess the efficiency oocyte enucleation assisted with PLM. In the first experiment, the presence of microtubule-polymerized protein was assessed and confirmed in oocytes (n = 117) by immunostaining and chromatin detection. In the second experiment, oocytes (n = 160) were exposed or not (controls) to PLM for 10 minutes, and then parthenogenetically activated and cultured in vitro. In the third experiment, development competence of oocytes with a positive or negative signal to PLM was analyzed after in vitro fertilization. Finally, oocytes (n = 54) were enucleated using PLM as a tool to remove the meiotic spindle. A positive PLM signal was detected in 98.2 % of the oocytes, which strongly correlated (r = 1; p < 0.0001) with the presence of microtubule-polymerized protein as confirmed by immunostaining. Oocytes exposed to PLM did not differ significantly from controls on cleavage, total blastocyst, expanded blastocyst rates and total cell numbers. The percentage of oocytes at the MII stage and blastocyst formation rate in the negative PLM group significantly differed from control and PLM positive groups. Overall efficiency of spindle removal using the PLM-Oosight system was 92.6%. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured porcine oocytes and does not exert detrimental effects on porcine oocyte developmental competence. Selecting oocytes by the presence of a PLM signal provides limited improvement on IVF results. Finally, PLM appears as an efficient method to enucleate porcine oocytes.

Introduction

The meiotic spindle plays a key role in normal chromosome alignment and segregation during meiosis. The meiotic spindles are assembled by microtubules, as their cytoskeletal components. Methods to detect and assess the meiotic spindles in oocytes have been described [1]. Compared with methods of spindle evaluation that require oocyte fixation, Polarized Light Microscopy (PLM) offers the unique advantage of being totally non-invasive, preserving oocyte viability and allowing repeated observations of the sample. PLM principles and equipment have been extensively described elsewhere [1], [2], [3], [4], [5]. The presence of the meiotic spindle appears to be associated with an increased fertilization rate and embryo quality in mouse and human [1], [6], [7], [8]. Interestingly, more oocytes with visible spindles fertilized and progressed to blastocysts compared to oocytes without visible spindles [9]. In addition, spindle retardance has been proposed as a marker of oocyte quality in human oocytes and it could be used to select oocytes with an improved embryonic developmental competence [10], [11], [12]. The structures and conformation of the meiotic spindles are important to predict the fate of the oocytes. If PLM is able to detect spindle abnormalities, it might become a valuable tool to select oocytes with normal spindle morphology and potentially higher developmental competence.

Within nuclear transfer, PLM can also be used as a tool to assist in the enucleation of oocytes. Several animal species have been successfully cloned by nuclear transfer [13], [14], [15], [16], [17], [18], [19], [20]. However, its overall efficiency is still very low [21], [22]. Enucleation is an important step towards a successful nuclear transfer and it can be accomplished by different methods such as blind removal of cytoplasm adjacent to the first polar body or by labelling the oocyte DNA with bisbenzimide (Hoechst 33342) and exposure to ultraviolet (UV) light to permit location and removal of chromosomes. Although the latter is a common practice in conventional somatic cell nuclear transfer (SCNT), Hoechst staining and UV light might be detrimental for oocyte developmental competence [23], [24], [25], [26].

Research in PLM has been carried out mainly in mice and human oocytes. However, information on the practical use of PLM in oocytes from farm animals, including pigs, is limited and its potential applications in animal reproduction need further assessment. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein in in vitro-matured porcine oocytes; to examine the effects of PLM on the oocyte developmental competence; to select oocytes based on the presence of the meiotic spindle detected by PLM and further assessment of their developmental competence; and to assess the efficiency of PLM as a tool to assist in the removal of the meiotic spindle of metaphase II oocytes.