Objective To confirm that oocyte-specific mRNAs are detectable in the polar body (PB) of MII oocytes and determine the effect of age on oocyte-specific transcript levels. Design Prospective study Setting Hospital-based academic research laboratory Animals CD1 female mice Intervention(s) Aged (40–50 weeks) and young (7–9 weeks) mice were administered pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG). Oocytes were fertilized in vitro to assess fertilization and developmental competence. MII oocytes were obtained and first PBs was removed. mRNA from each PB and its sibling oocyte was reverse transcribed and analyzed by real-time quantitative PCR. Main Outcome Measure(s) Fertilization and developmental rates and expression of 6 oocyte-specific genes (Bmp15, Gdf9, H1foo, Nlrp5, Tcl1, and Zp3) in PBs and sibling oocytes from young vs. aged mice. Result(s) Oocytes from aged mice had lower developmental competence. Four genes (H1foo, Nlrp5, Tcl1, and Zp3) were differentially expressed in aged vs. young oocytes (P < 0.05). All 6 transcripts were present in PBs from aged and young mice at lower levels than in the sibling oocytes; transcript levels were lower in aged PBs compared with young PBs (P < 0.05). Conclusion(s) There is a significant difference in the transcript levels of oocyte-specific genes in aged vs. young PB that correlates with age-related decreases in oocyte competence. Differences in gene expression in PB may be potential biomarkers of MII oocyte competence.

Objective To determine whether the follicle environment modulates oocyte-specific gene transcript levels in cultured oocytes and polar bodies (PBs). Design Animal study. Setting Large academic research center. Animal(s) CD1 mice. Intervention(s) In vitro growth of secondary mouse follicles in 0.25% or 1.5% alginate (ALG) in a three-dimensional culture system. Main Outcome Measure(s) Relative transcript levels of Gdf9, Bmp15, Nlrp5, Tcl1, and Zp3 were measured by real-time quantitative reverse transcriptase–polymerase chain reaction in oocytes during in vitro follicle development and oocyte maturation and in their first PBs after removal from metaphase II (MII) eggs. Result(s) All transcripts decreased earlier in oocytes cultured in 1.5% ALG compared with 0.25% ALG. Transcript levels were lower in MII eggs cultured in 1.5% ALG compared with in 0.25% ALG. All genes were expressed in PBs, and transcript levels were lower in PBs cultured in 1.5% ALG compared with in 0.25% ALG. Abundance of all transcripts was lower in PBs than in their sibling oocytes. Conclusion(s) Local follicle environment modulates oocyte-specific gene expression in the oocyte and first PB. There is a significant difference in the transcript levels of oocyte-specific genes in PBs of 1.5% versus 0.25% ALG that correlates with ovarian environment-related decreases in oocyte competence.

Objective To test the hypothesis that quantification of mRNAs originating the second polar body (PB2) provides a non-invasive tool for assessing embryo quality. Design Prospective study Setting Hospital-based academic research laboratory Animals CD1 female mice Intervention(s) MII oocytes obtained from 7- to 8-week-old mice after pregnant mare’s serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) priming. After in vitro fertilization, the PB2 was biopsied from zygotes followed by reverse transcription. Real-time PCR was performed to quantify gene expression levels in single PB2. The sibling zygotes were continuously cultured to blastocyst stage. Main Outcome Measure(s) Embryo developmental competence and six maternal-effect gene (Dnmt1, Mater, Nobox, Npm2, Tcl1 and Zar1) transcripts in the PB2 Results PB2 mRNA was detected in all candidate genes. Transcripts that were present in greater abundance in the zygote were more likely to be detected in qPCR replicates from single PB2. 4 candidate genes (Dnmt1, Nobox, Npm2 and Tcl1) expression levels in PB2 between two groups (2-cell embryo vs. blastocyts) approached statistical significance. Conclusion PB2 may contain a representative transcript profile to that of the zygote after fertilization. Differences in gene expression in PB2 may be potential biomarkers of embryo quality.

Purpose To confirm that aneuploidy candidate genes are detectable in the first polar body (PB 1 ) of MII oocytes and to investigate the age-dependent molecular changes in PB 1 . Methods Aged (12-to 15-mo-old) and young (2-mo-old) mice were administered pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG). MII oocytes were obtained and the first PB was removed. mRNA from each PB and its sibling oocyte was reverse transcribed. Real-time PCR was performed to quantify the expression of six genes (BUB1, CDC20, Filia, MCAK, SGOL1, SMC1A) in single PB. Results We first demonstrated that detection and quantification of transcripts associated with aneuploidy in single mouse oocyte and sibling PB 1 is possible and the relative abundance of mRNA transcripts in a single PB faithfully reflects the relative abundance of that transcript in its sibling oocyte. We further found that transcript levels were significantly lower in aged PBs compared with young PBs (P<0.05). Conclusions Our results suggest that the detection and analysis of polar body mRNA may provide insight in age-related aneuploidy in oocyte. This analysis is a novel concept to investigate the genesis of chromosome abnormality and could potentially assist in the characterization of mechanisms underlying key molecular origin of female meiotic aneuploidy, which would be of great scientific and clinical value.

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