The optimal decidualization of endometrial stromal cells (ESCs) following embryo implantation is one of the critical steps to establish pregnancy in rodents and humans. This step is intricately regulated by ovarian hormones. Using in vitro human ESCs model, we previously showed that activation of a cAMP mediator, exchange protein directly activated by cAMP (EPAC), promotes ovarian steroid- or cAMP analog-induced decidualization. However, expressions and functions of EPAC and RAP1 in the uterus during pregnancy have not yet been examined. In this study, we found that the expression of EPAC2 and RAP1 was markedly upregulated in the decidual cells at the implantation sites on days 7 and 9 of pregnancy in rats. Furthermore, both delayed-implantation and artificial decidualization models showed that EPAC2 and RAP1 expression was enhanced in decidual cells. Significant activation of cAMP-responsive element-binding protein (CREB), a central transcriptional factor of cAMP signaling, was observed in decidual cells. These spatiotemporal expressions of protein related EPAC pathway are overlapped by sites with activated cAMP signaling, indicating the association of EPAC signaling with decidualization. Strikingly, further studies in in vitro rat decidualization model showed that the cAMP analog and medroxyprogesterone stimulated the expression of decidual markers, while knockdown of EPAC1/2 and RAP1 attenuated the expressions of these markers. Together, these findings suggest that EPAC and RAP1 are the crucial factors for endometrial decidualization in rat pregnancy.
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- Abstract: placenta x
- Abstract: chorioallantoic x
- Abstract: trophoblast x
- Abstract: cytotrophoblast x
- Abstract: syncytiotrophoblast x
- Abstract: human placental lactogen x
- Abstract: syncytium x
- Abstract: decidualization x
- Abstract: decidua x
- Abstract: human chorionic gonadotropin x
- Abstract: trophectoderm x
- Abstract: embryo implantation x
Kazuya Kusama, Mikihiro Yoshie, Kazuhiro Tamura, Takiko Daikoku, Tsutomu Takarada and Eiichi Tachikawa
Hsiu-Wen Chan, Natalie C McKirdy, Hassendrini N Peiris, Gregory E Rice and Murray D Mitchell
Endocannabinoids are a family of lipid signalling molecules. As with prostaglandins (PGs), endocannabinoids are derived from polyunsaturated fatty acids and affect cell function via receptor-mediated mechanisms. They also bind to PG receptors, although at a lower affinity. The endocannabinoid network is regulated in pregnancy from embryo development to labour onset. Even small changes in endocannabinoid exposure can retard embryo development and affect implantation success. There is now compelling evidence that aberrant expression of factors involved in the endocannabinoid pathway in the placenta and circulating lymphocytes results in spontaneous miscarriage and poor pregnancy outcomes. It is likely that competition between endocannabinoids, PGs and other similar lipids ultimately determines how phospholipid/fatty acid substrates are metabolised and, thus, the balance between the uterotonic and tocolytic activities. We, therefore, hypothesise that endocannabinoid profiles may be used as a biomarker to predict and/or identify spontaneous labour onset.
GY Nie, AL Hampton, GQ Fu, YX Liu, JK Findlay and LA Salamonsen
Splicing factor SC35 is an essential component of the spliceosome, the cellular apparatus that removes introns from pre-mRNA to provide alternatively spliced isoforms. Many proteins associated with development of uterine receptivity and embryo implantation are present as isoforms, the tissue-specific expression of which may be regulated through alternative splicing. SC35 was identified as being increased at implantation sites during early pregnancy in mice. However, the present study has demonstrated that SC35 is present in human and rhesus monkey endometrium, that the protein is increased during the secretory phase of the oestrous cycle compared with the proliferative phase in both these primates and that it is present in a distinct pattern within the nucleus of both epithelial and stromal cells, as well as in cells of the vasculature. Both the intensity of immunoreactive protein and the proportion of cells that stain for SC35 alter with the phase of the oestrous cycle. A very precise expression pattern of SC35 (both protein and mRNA) was seen during early placentation in rhesus monkeys. At implantation sites between day 24 and day 35 of early pregnancy, SC35 was expressed strongly in cytotrophoblasts within the trophoblastic shell, in syncytiotrophoblast at the periphery of the cell column and in both cytotrophoblast and syncytiotrophoblast in the floating villi. In the adjacent maternal decidua, expression of SC35 was weak. These results indicate a role for SC35 in preparation of a receptive uterus, in the provision of secreted proteins to support blastocyst development and in trophoblast invasion.
D Hickford and L Selwood
This study describes for the first time the spatial and temporal distribution of a growth factor and its receptors in uteri and conceptuses of a marsupial species during the peri-gastrulation period. Uteri (gravid and non-gravid) and blastocysts from 40 female stripe-faced dunnarts (Sminthopsis macroura) were collected over the peri-gastrula period (days 6.0-8.5) and stained immunohistochemically for transforming growth factor beta2 (TGFbeta2) and its receptors, TbetaRI and TbetaRII, to determine possible roles for TGFbeta2 in marsupial embryonic development. The events that occurred during the period examined included the appearance and proliferation of hypoblast and mesoderm, primitive streak and node formation, and early neurulation. Differences in TGFbeta2 quantities between gravid and non-gravid uteri reflect differences in uterine morphology, indicating a role for TGFbeta2 in endometrial remodelling. In blastocysts, large quantities of all three proteins in the trophectoderm during the node stage coincide with both blastocyst expansion before implantation and the appearance of multinucleated cells, indicating that TGFbeta2 plays a role in conceptus elongation and trophectoderm differentiation. In contrast, lack of TbetaRII in blastocysts during hypoblast formation and proliferation negates any role for TGFbeta2 in these processes, as both receptors are required for a response to TGFbeta2. High concentrations of TGFbeta2 but low concentrations of TbetaRII in blastocysts during early primitive streak formation indicate that paracrinal embryo-maternal signalling may be occurring, as blastocysts cannot respond to TGFbeta2 at this stage, but uteri could. A similar situation, but reversed, also occurs during primitive node formation.
P. P. L. Tam, W. Y. Chan and H. W. Yeung
Summary. Mouse morulae and early blastocysts developed normally to the late blastocyst stage in the presence of α-momorcharin in culture. When these embryos were transferred to a pseudopregnant uterus, they showed a poor ability to induce the decidual reaction and many failed to implant. Those that had implanted showed retarded embryonic development and many implantation sites contained only trophoblastic giant cells and extraembryonic membranes. Implantation of blastocysts was inhibited when the recipient animal was given α-momorcharin at the time of embryo transfer. We suggest that termination of early pregnancy by α-momorcharin is the result of the deleterious effect of the protein on the implanting embryos and the endometrium.
Hong-Fei Xia, Jing-Li Cao, Xiao-Hua Jin and Xu Ma
MiR199a was found to be differentially expressed in rat uteri between the prereceptive and receptive phase via microRNA (miRNA) microarray analysis in our previous study. However, the role of miR199a in rat embryo implantation remained unknown. In the study, northern blot results showed that the expression levels of miR199a were higher on gestation days 5 and 6 (g.d.5–6) in rat uteri than on g.d.3–4 and g.d.7–8. In situ localization of miR199a in rat uteri showed that miR199a was mainly localized in the stroma or decidua. The expression of miR199a was not significantly different in the uteri of pseudopregnant rats and evidently increased in the uteri of rats subjected to activation of delayed implantation and experimentally induced decidualization. Treatment with 17β-estradiol or both 17β-estradiol and progesterone significantly diminished miR199a levels. Gain of function of miR199a in endometrial stromal cells isolated from rat uteri inhibited cell proliferation and promoted cell apoptosis. Loss of function of miR199a displayed opposite roles on cell proliferation and apoptosis. Further investigation uncovered a significant inverse association between the expression of miR199a and growth factor receptor-bound protein 10 (Grb10), an imprinted gene, and miR199a could bind to the 3′UTR of Grb10 to inhibit Grb10 translation. In addition, in vivo analysis found that the immunostaining of GRB10 was attenuated in the stroma or decidua from g.d.4 to 6, contrary to the enhancement of miR199a. Collectively, upregulation of miR199a in rat uterus during the receptive phase is regulated by blastocyst activation and uterine decidualization. Enforced miR199a expression suppresses cell proliferation partially through targeting Grb10.
I. Wilmut and D. I. Sales
Summary. Sheep embryos which were transferred to recipients in oestrus 3 days before or 3 days after the donors were unable to implant even when a synchronously transferred embryo was developing successfully in the same uterus. Embryos which had spent 3 days in an advanced recipient were transferred to a recipient synchronous with the original donor. Embryos first transferred on Day 3 were slightly accelerated in their development, but retained the ability to implant normally in the 2nd recipient. By contrast, embryos first transferred on Day 6 were markedly stimulated and less able to implant in the second recipient.
Michael J Boden, Tamara J Varcoe, Athena Voultsios and David J Kennaway
The light/dark cycle and suprachiasmatic nucleus rhythmicity are known to have important influences on reproductive function of rodents. We studied reproductive function in female heterozygous and homozygous brain and muscle ARNT-like protein 1 (Bmal1, also known as Arntl) null mice, which lack central and peripheral cellular rhythms. Heterozygous Bmal1 mice developed normally and were fertile, with apparent normal pregnancy progression and litter size, although postnatal mortality up to weaning was high (1.1–1.3/litter). The genotype distribution was skewed with both heterozygous and null genotypes underrepresented (1.0:1.7:0.7; P<0.05), suggesting loss of a single Bmal1 allele may impact on postnatal survival. Homozygous Bmal1 null mice were 30% lighter at weaning, and while they grew at a similar rate to the wild-type mice, they never achieved a comparable body weight. They had delayed vaginal opening (4 days), disrupted estrus cyclicity, and reduced ovarian weight (30%). Bmal1 null mice had a 40% reduction in ductal length and a 43% reduction in ductal branches in the mammary gland. Surprisingly, the Bmal1 mice ovulated, but progesterone synthesis was reduced in conjunction with altered corpora lutea formation. Pregnancy failed prior to implantation presumably due to poor embryo development. While Bmal1 null ovaries responded to pregnant mare serum gonadotropin/human chorionic gonadotropin stimulation, ovulation rate was reduced, and the fertilized oocytes progressed poorly to blastocysts and failed to implant. The loss of Bmal1 gene expression resulted in a loss of rhythmicity of many genes in the ovary and downregulation of Star. In conclusion, it is clear that the profound infertility of Bmal1 null mice is multifactorial.
Freda Wynne, Melanie Ball, Andrew S McLellan, Peter Dockery, Wolfgang Zimmermann and Tom Moore
The pregnancy-specific glycoproteins (Psg) are secreted hormones encoded by multiple genes in rodents and primates, and are thought to act as immune modulators. The only Psg receptor identified is CD9, through which Psg17 induces cytokine production from macrophages cultured in vitro. We examined temporal and spatial aspects of Psg and CD9 expression during mouse pregnancy to determine whether their expression patterns support a role in immune modulation. Using in situ hybridisation, immunohistochemistry and RT-PCR we found Psg expression in trophoblast giant cells and in the spongiotrophoblast. Psg22 is the predominant Psg family member expressed in giant cells. Detectable Psg is associated predominantly with endothelial cells lining vascular channels in the decidua, rather than with maternal immune cell markers. CD9 expression exhibited partial overlap with Psg, but without exclusive co-localisation. CD9 was observed in decidual cells surrounding early implantation sites, and in the endometrium. However, embryo transfer of wild-type embryos to CD9-deficient females indicates that maternal CD9 is not essential for successful pregnancy.
Thomas E Spencer, Greg A Johnson, Fuller W Bazer and Robert C Burghardt
Implantation in all mammals involves shedding of the zona pellucida, followed by orientation, apposition, attachment and adhesion of the blastocyst to the endometrium. Endometrial invasion does not occur in domestic ruminants; thus, definitive implantation is achieved by adhesion of the mononuclear trophoblast cells to the endometrial lumenal epithelium (LE) and formation of syncytia by the fusion of trophoblast binucleate cells with the LE. This review highlights new information on mechanisms regulating the implantation cascade in sheep. The embryo enters the uterus on day 4 at the morula stage of development and then develops into a blastocyst by day 6. The blastocyst sheds the zona pellucida (day 8), elongates to a filamentous form (days 11–16), and adheres to the endometrial LE (day 16). Between days 14 and 16, the binucleate cells begin to differentiate in the trophoblast and subsequently migrate and fuse with the endometrial LE to form syncytia. Continuous exposure of the endometrium to progesterone in early pregnancy downregulates the progesterone receptors in the epithelia, a process which is associated with loss of the cell-surface mucin MUC1 and induction of several secreted adhesion proteins. Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that secretions of the endometrial epithelia have a physiologic role in blastocyst elongation and implantation. A number of endometrial proteins have been identified as potential regulators of blastocyst development and implantation in sheep, including glycosylated cell adhesion molecule 1 (GlyCAM-1), galectin-15, integrins and osteopontin. The epithelial derived secreted adhesion proteins (GlyCAM-1, galectin-15 and osteopontin) are expressed in a dynamic temporal and spatial manner and regulated by progesterone and/or interferon tau, which is the pregnancy recognition signal produced by the trophoblast during blastocyst elongation. The noninvasive and protracted nature of implantation in domestic animals provides valuable opportunities to investigate fundamental processes of implantation that are shared among all mammals. Understanding of the cellular and molecular signals that regulate uterine receptivity and implantation can be used to diagnose and identify causes of recurrent pregnancy loss and to improve pregnancy outcome in domestic animals and humans.