ABSTRACT
Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.
Subject(s)
Embryonic Development/genetics , Ovary/metabolism , RGS Proteins/genetics , Animals , Blastocyst/metabolism , Cell Nucleus/metabolism , Female , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Male , Mice , RGS Proteins/metabolism , RNA, Small Interfering , Transcription Factors/genetics , Transcription Factors/metabolism , Zygote/metabolismABSTRACT
BACKGROUND: This study was conducted to observe the in vivo effect of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) on embryo implantation in rats and its in vitro effect on cell adhesion. STUDY DESIGN: The anti-implantation efficacy of AEBSF in rats was determined by counting the number of visible implanted embryos on day 8 of pregnancy following intrauterine (5 mg and 10 mg AEBSF per horn) or tail vein (10 mg AEBSF per rat) administration on day 3 of pregnancy. The effects of AEBSF on cell adhesion were detected, respectively, by using the mouse blastocysts-endometrial cells or the human umbilical vein endothelial cells (HUVECs)-HeLa cells co-culture model. The alteration in protein secretion pattern of HUVECs and HeLa cells was detected by the proteome analysis. RESULTS: 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride showed an in vivo inhibitory effect on embryo implantation in rat. In vitro, AEBSF could disturb the growth of blastocysts on endometrial cells and inhibit the adhesion of HeLa cells on HUVECs. The treatment of AEBSF could alter the protein secretion pattern of co-cultured HUVEC-HeLa cells. CONCLUSION: 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride might be a potential leading compound for novel contraceptives, and its inhibitory effect on implantation might result from the interference in extracellular matrix remodeling process.
Subject(s)
Embryo Implantation/drug effects , Serine Proteinase Inhibitors/pharmacology , Sulfones/pharmacology , Administration, Intravaginal , Animals , Blastocyst/drug effects , Cell Adhesion/drug effects , Cells, Cultured , Coculture Techniques , Contraceptives, Postcoital, Synthetic/administration & dosage , Contraceptives, Postcoital, Synthetic/pharmacology , Dose-Response Relationship, Drug , Endometrium/cytology , Endometrium/drug effects , Female , HeLa Cells , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Injections, Intravenous , Mice , Mice, Inbred ICR , Pregnancy , Random Allocation , Rats , Rats, Sprague-Dawley , Serine Proteinase Inhibitors/administration & dosage , Sulfones/administration & dosageABSTRACT
To investigate the biological significance of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) involvement in oocyte maturation, we screened for proteins that bound to UCH-L1 in mouse ovaries, and we found that the prostate tumor overexpressed-1 (PTOV1) protein was able to bind to UCH-L1. PTOV1 is highly expressed in prostate cancers and considered as a potential marker for carcinogenesis and the progress of prostate cancer. It was reported that PTOV1 plays an important role in cell cycle regulation, but its role in mammalian oocyte development and meiosis is still unclear. In this paper, it was found that the expression levels of PTOV1 in mouse ovaries progressively increased from prepubescence to adulthood. And we found by immunohistochemistry that PTOV1 spreaded in both the cytoplasm and nuclei of oocytes during prepuberty, but in normal adult mouse oocytes, it concentrated not only in nuclei but also on the plasma membrane, though in some oocytes with abnormal shapes, PTOV1 did not display the typical distribution patterns. In granulosa cells, however, it was found to locate in the cytoplasm at all the selected ages. In postnatal mouse ovaries (28 days), estradiol treatment induced the adult-specific distribution pattern of PTOV1 in oocytes. In addition, UCH-L1 was shown to be associated with CDK1, which participated in the regulation of cell cycle and oocyte maturation. Therefore, we propose that the distribution changes of PTOV1 are age-dependent, and significant for mouse oocyte development and maturation. Moreover, the discovery that PTOV1 is associated with UCH-L1 in mouse oocytes supports the explanations for that UCH-L1 is involved in oocyte development and maturation, especially under the regulation of estrogen.
