Zeng-Ming Yang

Differential Expression of Microsomal Prostaglandin E Synthase at Implantation Sites and in Decidual Cells of Mouse Uterus

Abstract Prostaglandin E2 (PGE2) is considered important for blastocyst spacing, implantation, and decidualization in the rodent uterus. PGE synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. There are two isoforms of PGES, microsomal PGES (mPGES) and cytosolic PGES (cPGES). However, the expression and regulation of mPGES in the mammalian uterus during early pregnancy are still unknown. The aim of this study was to investigate the differential expression of mPGES in mouse uterus during early pregnancy and its regulation under different conditions by in situ hybridization and immunohistochemistry. Microsomal PGES expression in the preimplantation mouse embryos was also performed by reverse transcription polymerase chain reaction (RT-PCR). Expression of mPGES mRNA and protein was at a basal level in the luminal epithelium from Day 1 to Day 4 of pregnancy. However, mPGES mRNA and protein were highly expressed in the stroma immediately surrounding the blastocyst but not in the luminal epithelium on Day 5 of pregnancy. Microsomal PGES mRNA and protein were not detected in the pseudopregnant uterus from Day 1 to Day 5. During delayed implantation, mPGES mRNA and protein were also not detected in the uterus. Once delayed implantation was terminated by estrogen treatment and embryo implantation initiated, both mPGES mRNA and protein were induced to express in the stroma immediately surrounding the blastocyst, which was similar to the expression pattern on Day 5 of pregnancy. From Day 6 to Day 8 of pregnancy, the signals for mPGES mRNA and protein were strongly detected in the decidualized cells. Microsomal PGES mRNA and protein were also highly expressed in the artificially decidualized cells but not in the control horn. Microsomal PGES mRNA was detected in the oocytes and all the stages of preimplantation embryos. The strong mPGES expression in the implantation site and decidual cells suggests that mPGES might play an important role during implantation and more importantly in decidualization.

Leukemia Inhibitory Factor, Leukemia Inhibitory Factor Receptor, and Glycoprotein 130 in Rhesus Monkey Uterus During Menstrual Cycle and Early Pregnancy

Abstract This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.

Polyamines are essential in embryo implantation: Expression and function of polyamine-related genes in mouse uterus during peri-implantation period

Polyamines are key regulators in cell growth and differentiation. It has been shown that ornithine decarboxylase (Odc) was essential for post-implantation embryo development, and overexpression of spermidine/spermine N1-acetyltransferase will lead to ovarian hypofunction and hypoplastic uteri. However, the expression and function of polyamine-related genes in mouse uterus during early pregnancy are still unknown. In this study we investigated the expression, regulation, and function of polyamine-related genes in mouse uterus during the peri-implantation period. Odc expression was strongly detected at implantation sites and stimulated by estrogen treatment. The expression of Odc antizyme 1 and spermidine/spermine N1-acetyltransferase was also highly shown at implantation sites and regulated by Odc or polyamine level in uterine cells. Embryo implantation was significantly inhibited by alpha-difluoromethylornithine, an Odc inhibitor. Moreover, the reduction of Odc activity caused by alpha-difluoromethylornithine treatment was compensated by the up-regulation of S-adenosylmethionine decarboxylase gene expression. Collectively, our results indicated that the coordinated expression of uterine polyamine-related genes may be important for embryo implantation.

The Integrative Analysis of microRNA and mRNA Expression in Mouse Uterus under Delayed Implantation and Activation

Background Delayed implantation is a developmental arrest at the blastocyst stage and a good model for embryo implantation. MicroRNAs (miRNAs) have been shown to be involved in mouse embryo implantation through regulating uterine gene expression. This study was to have an integrative analysis on global miRNA and mRNA expression in mouse uterus under delayed implantation and activation through Illumina sequencing. Methodology/Principal Findings By deep sequencing and analysis, we found that there are 20 miRNAs up-regulated and 42 miRNAs down-regulated at least 1.2 folds, and 268 genes up-regulated and 295 genes down-regulated at least 2 folds under activation compared to delayed implantation, respectively. Many different forms of editing in mature miRNAs are detected. The percentage of editing at positions 4 and 5 of mature miRNAs is significantly higher under delayed implantation than under activation. Although the number of miR-21 reference sequence under activation is slightly lower than that under delayed implantation, the total level of miR-21 under activation is higher than that under delayed implantation. Six novel miRNAs are predicted and confirmed. The target genes of significantly up-regulated miRNAs under activation are significantly enriched. Conclusions miRNA and mRNA expression patterns are closely related. The target genes of up-regulated miRNAs are significantly enriched. A high level of editing at positions 4 and 5 of mature miRNAs is detected under delayed implantation than under activation. Our data should be valuable for future study on delayed implantation.

Estrogen Regulates Amiloride-Binding Protein 1 through CCAAT/Enhancer-Binding Protein-β in Mouse Uterus during Embryo Implantation and Decidualization

Embryo implantation is an intricate interaction between receptive uterus and active blastocyst. The mechanism underlying embryo implantation is still unknown. Although histamine and putrescine are important for embryo implantation and decidualization, excess amount of histamine and putrescine is harmful. Amiloride binding protein 1 (Abp1) is a membrane-associated amine oxidase and mainly metabolizes histamine and putrescine. In this study, we first showed that Abp1 is strongly expressed in the decidua on d 5-8 of pregnancy. Abp1 expression is not detected during pseudopregnancy and under delayed implantation but is detected after estrogen activation. Because Abp1 is mainly localized in the decidua and also strongly expressed during in vitro decidualization, Abp1 might play a role during mouse decidualization. The regulation of estrogen on Abp1 is mediated by transcription factor CCAAT/enhancer-binding protein-β. Abp1 expression is also regulated by cAMP, bone morphogenetic protein 2, and ERK1/2. Abp1 may be essential for mouse embryo implantation and decidualization.

Basigin expression and hormonal regulation in mouse uterus during the peri‐implantation period

Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, has been shown to be essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of basigin gene in mouse uterus during the peri‐implantation period. Basigin immunostaining and mRNA were strongly localized in luminal and glandular epithelium on day 1 of pregnancy and gradually decreased to a basal level from day 2–4 of pregnancy. Basigin mRNA expression in the sub‐luminal stroma was first detected on day 3 of pregnancy and increased on day 4 of pregnancy. On day 5 of pregnancy, the expression of basigin protein and mRNA was only detected in the implanting embryos, and the luminal epithelium and sub‐luminal stroma surrounding the embryos. A similar expression pattern of basigin was also induced in the delayed‐implantation uterus which was activated by estrogen injection. On day 6–8 of pregnancy, although a basal level of basigin protein was detected in the secondary decidual zone, basigin mRNA expression was strongly seen in this location. Basigin mRNA was also highly expressed in the decidualized cells under artificial decidualization. Estrogen significantly stimulated basigin expression in the ovariectomized mouse uterus. A high level of basigin immunostaining and mRNA was also seen in proestrus and estrus uteri. These results suggest that basigin expression is closely related to mouse implantation and up‐regulated by estrogen. Mol. Reprod. Dev. 63: 47–54, 2002.

Effects of platelet activating factor on capacitation and acrosome reaction in mouse spermatozoa

Platelet activating factor (PAF) plays an important role in mammalian reproduction. The aim of this study was to investigate the effects of PAF on capacitation and acrosome reaction of mouse spermatozoa by chlortetracycline (CTC) fluorescence assay and coomassie blue staining. The percentage of capacitated mouse spermatozoa was increased (P < 0.05) by incubation with 50 ng/ml PAF for 20–120 min. The peak response occurred between 80 to 100 min of exposure to PAF. In contrast, the effects of PAF on acrosome reaction may be not receptor‐mediated since lyso‐PAF had the same effects. Ionophore A23187 stimulated an increase in acrosome‐reacted spermatozoa of PAF‐treated spermatozoa, but not of lyso‐PAF‐treated ones. These results suggest that PAF mainly acts on sperm capacitation. Mol. Reprod. Dev. 56:436–440, 2000.

Epidermal growth factor family in rhesus monkey uterus during the menstrual cycle and early pregnancy.

This study examines immunohistochemically the presence of EGF, TGFα, HB‐EGF, AR, and EGFR, members of the EGF family in the monkey uterus during the menstrual cycle and early pregnancy. EGF, TGFα, HB‐EGF, AR, and EGFR were mainly localized in glandular and luminal epithelium. TGFα, HB‐EGF, and AR staining were stronger in the glandular epithelium closer to the myometrium than in that closer to the luminal epithelium. The level of EGF, TGFα, HB‐EGF, AR, and EGFR staining was low on days 1 and 6, and began to increase on day 9 of the menstrual cycle. A high level of EGF, and EGFR staining was maintained on days 16, 20, and 25 of the menstrual cycle. The highest levels of TGFα, AR, and HB‐EGF staining were seen on days 16 and 20 of the menstrual cycle. In early pregnancy, a low level of EGF, TGFα, HB‐EGF, AR, and EGFR staining appeared on days 1 and 2 of pregnancy, and then gradually increased from day 3 of pregnancy. The highest levels of EGF, TGFα, HB‐EGF, and EGFR were detected on days 9, and 11 of pregnancy. Our data suggest that the EGF family may play a role in monkey implantation. Mol. Reprod. Dev. 55:164–174, 2000.

Expression and Regulation of Lipocalin-Type Prostaglandin D Synthase in Rat Testis and Epididymis

Abstract Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.

Rat ovulation, implantation and decidualization are severely compromised by COX-2 inhibitors.

Although Cyclooxygenase-2 (COX-2) is essential for mouse ovulation, fertilization, implantation and decidualization, the regulation and function of COX-2 in rat reproduction are still unknown. This study was designed to examine the action of COX-2 in rat ovulation, implantation and decidualization by using two specific inhibitors of COX-2 (nimesulide and niflumic acid). Compared to control, either nimesulide or niflumic acid significantly inhibited the ovulation in the superovulated rats. Although nimesulide had no obvious effects on the number of implantation sites and the vascular permeability, the expression of PPARdelta, HB-EGF and vimentin proteins was down-regulated in the nimesulide-treated groups. COX-1 protein was upregulated by nimesulide treatment. Nimesulide also had an inhibitory effect on decidualization during early pregnancy and under artificial decidualization. Moreover, nimesulide caused the increase of the gestation period and the reduction of litter size and birth weight compared to controls. Based on our data, rat implantation and decidualization were delayed by nimesulide treatment, resulting in the reduction of litter size and birth weight and the prolongation of gestational length, suggesting that COX-2 plays an important role in implantation and decidualization.