A Delayed-Implantation-Associated Protein Prevents Resumption of DNA Synthesis by the Trophectoderm of Delayed-Implanting Mouse Blastocysts in vitro.

Abstract

ABSTRACT. Objective of this study was to determine the ability of a delayed-implantation-associated protein (MW 170,000, DIAP170K) to inhibit DNA synthesis by mouse blastocysts. Mice were ovariectomized on day 3 of pregnancy and treated with daily injections with 1 mg progesterone till day 7 to induce delayed implantation. Blastocysts were collected on day 8 with or without a single injection of 25 ng estradiol-17β on day 7 that activates blastocyst metabolisms (activated blastocysts and delayed-implanting blastocysts respectively). DNA synthesis was determined by measuring [ 3 H]thymidine incorportion by blastocysts. DIAP170K at 10 µg/ml suppressed resumption of DNA synthesis by delayed-implanting blastocysts and suppression was maximal at 50 µg/ml. However, DIAP170K did not affect DNA synthesis by blastocysts obtained on day 5 of pregnancy (normal blastocysts) and activated blastocysts. Resumption of DNA synthesis in the inner cell mass (ICM) and trophectoderm from delayed-implanting blastocysts was then separately assessed. DNA synthesis resumed in the trophectoderm of intact blastocysts during 24-hr culture but not in the trophectoderm cultured apart from the ICM. DIAP170K inhibited the resumption of DNA synthesis by the trophectoderm of intact delayed-implanting blastocysts but did not affect DNA synthesis by the ICM. In conclusion, DIAP170K inhibits resumption of DNA synthesis by trophectoderm of delayed-implanting blastocysts. This action of DIAP170K may play a central role in maintaining, but not achieving, dormancy of DNA synthesis by delayed-implanting blastocysts in mice. MATERIALS AND METHODSBlastocysts: Virgin 8-12 week-old CD1 female mice (Animal Care Centre, University of British Columbia, Vancouver, Canada) were housed with fertile males of the same strain overnight on the day of estrus. Females with a vaginal plug on the following morning were considered pregnant (day 1). Females were randomly divided into 3 groups. Mice in the first group were bilaterally ovariectomized on day 3 and given daily injections (s.c.) with 1 mg progesterone (Sigma, St. Louis, U.S.A.) prepared in 0.05 ml sesame oil until day 7. Blastocysts were collected on day 8 (delayed-implanting blastocysts). Mice in the second group were ovariectomized, followed by daily progesterone injections as above plus a single injection (s.c.) of 25 ng estradiol-17β (Sigma) in 0.05 ml sesame oil on day 7. Blastocysts were collected on day 8 (activated blastocysts). Mice in the last group were intact and blastocysts were collected on day 5 (normal blastocysts).DIAP170K preparation: DIAP170K was obtained from mouse uteri during experimentally induced delayed implantation [7]. Partially purified DIAP170K used in this study showed one broad band (>97% of total protein by HPLC analysis) and a few trace bands on SDS-PAGE under reduced and unreduced conditions. The apparent molecular weight of the main band was estimated as 168,500 on SDS-PAGE.Blastocyst culture: Blastocysts were cultured in DNA synthesis as well as other metabolic events in mou...

Involvement of microRNA Lethal-7a in the Regulation of Embryo Implantation in Mice

“…Dormancy is associated with reduction in DNA synthesis, cell cycling and metabolism [29] , [30] . In the delayed implantation mouse model, estrogen-induced activation stimulates mitosis and increases cell number of the dormant blastocysts in vivo [31] , consistent with the predicted involvement of the potential target genes of the differentially expressed miRNAs in cell cycle, nucleic acid metabolism and metabolic processes.…”

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“…The 170-kDa delayed-implantation-associated protein (DIAP 170K) was found in the uterine tissue as well as in the uterine luminal fluid. The abilities of this DIAP 170K to suppress RNA synthesis and cell proliferation as well as to suppress resumption of DNA synthesis by delayed blastocysts may play a role in the regulation of embryonic dormancy associated with delayed implantation (16,17).…”