Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice

Abstract

Errors in meiotic chromosome segregation are the leading cause of spontaneous abortions and birth defects. Almost all such aneuploidy derives from meiotic errors in females, with increasing maternal age representing a major risk factor. It was recently reported that histones are globally deacetylated in mammalian oocytes during meiosis but not mitosis. In the present study, inhibition of meiotic histone deacetylation was found to induce aneuploidy in fertilized mouse oocytes, which resulted in embryonic death in utero at an early stage of development. In addition, a histone remained acetylated in the oocytes of older (10-month-old) female mice, suggesting that the function for histone deacetylation is decreased in the oocytes of such mice. Thus, histone deacetylation may be involved in the fair distribution of chromosomes during meiotic division. The high incidence of aneuploidy in the embryos of older females may be due to inadequate meiotic histone deacetylation.

Fig. 1.

Inhibition of histone deacetylation during meiotic maturation by TSA. GV-stage oocytes (GV) were cultured in vitro with (TSA+) or without (TSA−) TSA and allowed to undergo meiotic maturation. Eighteen hours after isolation from the follicles, at which time the oocytes had reached the MII stage, the oocytes were collected and immunostained with Ab against acetylated H4K12 (AcH4K12). The Ab was localized with FITC-conjugated secondary Ab (green), and DNA was stained with DAPI (blue). The experiment was performed three times with similar results; representative examples are shown.

Fig. 2.

Inhibition of histone deacetylation during meiosis affects embryo survival. (A) Two-cell stage embryos treated with TSA during meiosis were transferred into pseudopregnant females (TSA+), and the number of pups born was counted. As a control, two-cell-stage embryos treated with TSA only at fertilization were transferred into pseudopregnant females (TSA−). For each treatment, we used seven pseudopregnant females and transferred 7–11 embryos per oviduct. Females transplanted with TSA-treated embryos delivered significantly fewer offspring than females transplanted with control embryos (P < 0.005, Student’s t test). (B) Females transplanted with TSA-treated (TSA+) or control (TSA−) embryos were killed at 12 days postcoitus. Three or more pseudopregnant females were used for each treatment, and 10 embryos per oviduct were transferred. Embryo resorption in females transplanted with TSA-treated embryos was significantly higher than in females transplanted with control embryos (P < 0.05, Student’s t test). Data represent the mean ± SE.

Fig. 3.

One-cell zygotes treated with TSA during meiosis showed chromosomal aneuploidy. Oocytes were treated with TSA from the GV stage until 3 h after insemination (TSA+) or only immediately after insemination until 3 h later (TSA−). (A) Zygotes at the one-cell stage were mitotically arrested by colcemid treatment, and the chromosomal karyotypes were examined. Individual pronuclei are indicated by dotted lines; the arrow indicates a pronucleus with one extra chromosome (n = 21 chromosomes). (B) We analyzed the number of chromosomes at the one-cell stage. One-cell zygotes treated with TSA during meiotic maturation showed a significantly higher incidence of aneuploidy (the sum of hypo- and hyperploidy) compared with control zygotes (P < 0.01, χ² test). (C) TSA treatment caused aberrant chromosomal arrangement in MII-stage oocytes. GV-stage oocytes were cultured in vitro with (TSA+) or without (TSA−) TSA. The oocytes that had reached the MII stage were immunostained with an Ab against acetylated H4K12 (AcH4K12, green) and α-tubulin (red), and the DNA was stained with DAPI (blue).

Fig. 4.

Histone acetylation persists in MII-stage oocytes from older mice. (A) MII-stage oocytes obtained from young (3 weeks) and older (10 months) female mice were immunostained with Ab against acetylated H4K12 (AcH4K12). The Ab was localized with FITC-conjugated secondary Ab (green), and DNA was stained with DAPI (blue). (B) Histone acetylation was completely undetectable in all oocytes from young mice but was frequently observed in the oocytes from old mice.