Detection and quantification of mRNA in single human polar bodies: A minimally invasive test of gene expression during oogenesis

Division of Reproductive Endocrinology and Infertility, Women and Infants Hospital, Alpert School of Medicine, Brown University, 101 Dudley Street, Providence, RI 02905, USA.
Molecular Human Reproduction (Impact Factor: 3.75). 12/2010; 16(12):938-43. DOI: 10.1093/molehr/gaq077
Source: PubMed


Proteins and mRNA produced in oogenesis support embryonic development until the zygotic transition, 3 days after fertilization.
Since polar bodies can be biopsied with little if any harm to the oocyte, we tested the hypothesis that mRNA originating from
expression in the meiotic oocyte is present and detectable in a single polar body prior to insemination. Human oocytes were
obtained from patients undergoing controlled ovarian hyperstimulation and intracytoplasmic sperm injection. Immature oocytes
were cultured overnight and inspected the following day for maturation. Metaphase II oocytes underwent polar body biopsy followed
by reverse transcription without RNA isolation. Sibling oocytes were similarly prepared. Complementary DNA from all samples
were pre-amplified over 15 cycles for candidate genes using selective primers. Real-time PCR was performed to detect and quantify
relative single-cell gene expression. Polar body mRNA was detected in 11 of 12 candidate genes. Transcripts that were present
in greater abundance in the oocyte were more likely to be detected in qPCR replicates from single polar bodies. Pre-amplification
of cDNA synthesized without RNA isolation can facilitate the quantitative detection of mRNA in single human polar bodies.

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    • "in the meiotic MII oocyte from the in vitro fertilization programme is present and detectable in a single polar body prior to fertilization [30]. In their study immature oocytes from the intracytoplasmic sperm injection (ICSI) programme were cultured overnight and checked on the following day for their maturity. "
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    ABSTRACT: The quality of the human oocyte determines the success of fertilization and affects the consequent embryo development, pregnancy and birth; it therefore serves as a basis for human reproduction and fertility. The possibility to evaluate oocyte quality in the in vitro fertilization programme is very limited. The only criterion which is commonly used to evaluate oocyte quality is its morphology. There is a mass of oocytes in the in vitro fertilization programme which are not fertilized in spite of normal morphology. In the past, several attempts focused on oocyte gene expression profiling by different approaches. The results elucidated groups of genes related to the human oocyte. It was confirmed that some factors, such as oocyte in vitro maturation, are detectable at the molecular level of human oocytes and their polar bodies in terms of gene expression profile. Furthermore, the first genetic evaluations of oocyte-like cells developed in vitro from human stem cells of different origin were performed showing that these cells express some genes related to oocytes. All these findings provide some new knowledge and clearer insights into oocyte quality and oogenesis that might be introduced into clinical practice in the future.
    Full-text · Article · Feb 2013
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    • "Recent published literature suggests that discovery of novel transcripts in oocytes is still an open research agenda (Klatsky et al., 2010). Continued identification of new oocyte-specific genes and their functional annotation is providing additional insight into the molecular mechanism involved in folliculogenesis, oogenesis, fertilization and early embryogenesis (Bettegowda et al., 2007; Wells et al., 2008; Luo et al., 2010). "
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    ABSTRACT: A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.
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