Se-Jin Yoon

Leptin receptors are down-regulated in uterine implantation sites compared to interimplantation sites

Leptin is a circulating hormone that plays an important role in the regulation of metabolism, obesity, and reproduction. Leptin binds to its receptors on the cell membrane and is involved in the activation of STAT3. Recently, endometrium was suggested to be a novel target for leptin recently. We, therefore, examined the expression of leptin, leptin receptors, and STAT3 in the mouse uterus (implantation and interimplantation sites) to investigate the role of the leptin system during the early implantation period. Leptin mRNA was not detected in mouse uterine tissues or blastocysts, although adipose tissue, the positive control, showed a strong signal. Both of the receptor splice variants were expressed in the uterus and blastocysts, but the mRNA level was much lower in implantation sites compared to interimplantation sites. The mRNA expression of leptin receptors was determined to be higher in stromal cells than in the luminal epithelium using laser capture microdissection (LCM) analysis. Using immunohistochemistry, leptin was detected as a strong signal in the luminal epithelium and embryo, whereas the receptor was detected in subepithelial stromal cells rather than the luminal epithelium. As leptin itself was not detected by RT-PCR, the immunohistologically detected leptin may originate elsewhere, such as in adipose tissue. The differential expression of leptin receptors in implantation sites compared to interimplantation sites suggests that the leptin/leptin receptor system may be a delicate regulator of the implantation process.

Role of Bcl2-like 10 (Bcl2l10) in Regulating Mouse Oocyte Maturation

Previously, we have shown that Bcl2l10 is highly expressed in metaphase II (MII)-stage oocytes. The objective of this study was to characterize Bcl2l10 expression in ovaries and to examine the function of Bcl2l10 in oocyte maturation using RNA interference. Bcl2l10 transcript expression was ovary and oocyte specific. Bcl2l10 was highly expressed in oocytes and pronuclear-stage embryos; however, its expression decreased at the two-cell stage and dramatically disappeared thereafter. Microinjection of Bcl2l10 double-stranded RNA into the cytoplasm of germinal vesicle oocytes resulted in a marked decrease in Bcl2l10 mRNA and protein and metaphase I (MI) arrest (78.9%). Most MI-arrested oocytes exhibited abnormalities in their spindles and chromosome configurations. Bcl2l10 RNA interference had an obvious effect on the activity of maturation-promoting factor but not on that of mitogen-activated protein kinase. We concluded that the role of Bcl2l10 is strongly associated with oocyte maturation, especially at the MI–MII transition.

A molecular basis for embryo apposition at the luminal epithelium

To obtain a gene expression profile during embryo apposition to the luminal epithelium, we isolated mouse luminal epithelium from implantation (IM) and interimplantation (INTER) sites using laser capture microdissection (LCM), and analyzed their gene expression by microarray analysis. IM and INTER sites were sampled on day 4.5 after mating of female mice with fertile males (day 0.5 = vaginal plug). RNA was extracted, amplified, labeled, and hybridized to microarrays and results were analyzed using the significance analysis of microarrays (SAM) method. Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes most highly expressed at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were ESTs or were of unknown function, and the remain 53 genes had known functions mainly relating to cellular structuring and others such as cell cycling, gene/protein expression, immune responses, invasion, metabolism, oxidative stress, or signal transduction. Specifically, of the 24 structural genes, 14 were implicated in extracellular matrix and tissue remodeling. Meanwhile, of the 13 genes that were highly expressed at INTER, eight were ESTs or of unknown function, and the remaining five were implicated in metabolism, signal transduction, and gene/protein expression. Among these 58 (53 + 5) genes with known functions, 13 genes (22.4%) were associated with Ca2+ for their function. Results of the present study suggest that (1) at IM sites, active tissue remodeling is occurring for embryo invasion while the INTER sites are relatively quiescent and (2) Ca2+ may be a vital regulatory factor in the apposition process. Investigations of human homologues of those genes expressed in the mouse luminal epithelium during apposition may help to understand the implantation process and/or implantation failure in humans.

Function of COP9 signalosome in regulation of mouse oocytes meiosis by regulating MPF activity and securing degradation.

The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C.

Role of Bcl2l10 in Regulation of Meiotic Cell Cycle in the Mouse Oocyte

The pro-apoptotic factor Bad is expressed in various tissues including rat ovaries and testes (Kaipia et al., 1997). Bok, anti-apoptotic factor, has been detected in granulosa cells as well as in several reproductive tissues such as the ovaries, testes, and uterus (Hsu et al., 1997). Several anti-apoptotic Bcl2 homologs are expressed in the ovary. Mcl1, Bcl2, Bcl2l1 (Bcl-x), and other Bcl2 family members have also been detected in ovarian tissues (Hsu et al., 1997; Hsu and Hsueh, 2000; Kim and Tilly, 2004; Tilly et al., 1995). However, the expression patterns of these Bcl2 family members are quite different from those of Bcl2l10, which exhibits ovaryand oocyte-specific expression. Murine Bcl2l10 was first identified in expressed sequence tag clones from unfertilized, fertilized, and two-cell-stage mouse eggs (Inohara et al., 1998). Expression of this factor is restricted to the ovary and testis in adult mice (Inohara et al., 1998).