The mouse embryo autonomously acquires anterior-posterior polarity at implantation

The earliest recognizable sign of patterning of the mouse embryo along the anteroposterior (A-P) axis is the migration of the distal visceral endoderm (DVE) toward the future anterior side. Here we report an asymmetry in the mouse embryo at an unexpectedly early stage. The gene for Lefty1, a Nodal antagonist that influences the direction of DVE migration, was found to be asymmetrically expressed in the primitive endoderm of the implanting blastocyst. Lefty1 expression begins randomly in the inner cell mass (ICM) of the blastocyst but is regionalized to one side of the tilted ICM shortly after implantation. Asymmetric expression of Lefty1 can be established by in vitro culture, indicating that it does not require interaction with the uterus. The asymmetric Lefty1 expression is induced by Nodal signaling, although Nodal and genes for its effectors are expressed symmetrically. This asymmetry in molecular patterning of the mouse embryo pushes back the origin of the A-P body axis to the peri-implantation stage.     

Morphology of the mouse embryo,from the time of implantation to mesoderm formation

Summary The preparation technique of electron microscopy was adapted to light microscopy, in attempts to obtain well preserved implantation sites. The most appropriate technique comprised perfusion fixation in glutaraldehyde, post-fixation in osmium acid, Epon-embedding, ultramicrotomy, and staining with toluidine blue.The morphology of the early mouse embryo from the time of nidation to mesoderm formation is described: the formation of Reichert's membrane occurs already at 6 1/2 days, by which time free trophoblast cells are to be found in the uterine cavity.     

生命过程的相似性--从着床部位母体细胞的凋亡谈起

胚胎着床受许多因素的精确调控,其中着床部位母体细胞的凋亡在围植入期执行着重要的生理任务。但它自身的发生机制尚不完全清楚,在胚胎着床中的调节机制就更远远落后于凋亡在其他系统领域中的相知程度。因此,对细胞凋亡在着床部位母体细胞中出现的深入研究,将进一步完善我们对着床机制的理解,同时,因为肿瘤和胚胎对机体作用相似性也使这个领域的研究具有特殊的意义。     

Effects of anandamide on embryo implantation in the mouse

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that 14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R. However, 56 nM anandamide is capable of inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that anandamide may play an essential role at the outset of implantation.     

Polarity of the mouse embryo is anticipated before implantation

In most species, the polarity of an embryo underlies the future body plan and is determined from that of the zygote. However, mammals are thought to be an exception to this; in the mouse, polarity is generally thought to develop significantly later, only after implantation. It has not been possible, however, to relate the polarity of the preimplantation mouse embryo to that of the later conceptus due to the lack of markers that endure long enough to follow lineages through implantation. To test whether early developmental events could provide cues that predict the axes of the postimplantation embryo, we have used the strategy of injecting mRNA encoding an enduring marker to trace the progeny of inner cell mass cells into the postimplantation visceral endoderm. This tissue, although it has an extraembryonic fate, plays a role in axis determination in adjacent embryonic tissue. We found that visceral endoderm cells that originated near the polar body (a marker of the blastocyst axis of symmetry) generally became distal as the egg cylinder formed, while those that originated opposite the polar body tended to become proximal. It follows that, in normal development, bilateral symmetry of the mouse blastocyst anticipates the polarity of the later conceptus. Moreover, our results show that transformation of the blastocyst axis of symmetry into the axes of the postimplantation conceptus involves asymmetric visceral endoderm cell movement. Therefore, even if the definitive axes of the mouse embryo become irreversibly established only after implantation, this polarity can be traced back to events before implantation.     

Uterine estrogen sulfatase activity at the time of blastocyst implantation in the rat

The conversion of estrone sulfate (E1S) to estrone (E1) was measured during the in vitro incubation of the labeled sulfoconjugate with implantation sites (IS) and nonimplanted regions (NIS) of uterine horns from 6-day pregnant rats. Extensive metabolism of E1S occurred in both tissues, being noticeably less (29.31%) in IS than in NIS. Estrogen sulfatase activity present in the uterus of ovariectomized virgin rats was found to be higher than in both uterine regions of the pregnant rats. We suggest that E1S present in uterine fluids may be accessible to be metabolized into unconjugated estrogens by both intrauterine tissues of 6-day pregnant rats. This metabolism could be locally modulated in IS through the participation of the estrogen sulfatase, the activity of which is in turn controlled by the presence of free estrogens, possibly synthesized and/or secreted by the embryo, which has been shown to inhibit the sulfohydrolase activity.     

Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture.

Cell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus. The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow. We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM). In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL [Terminal deoxynucleotidyl transferase mediated d-UTP nick end-labelling] labelling at the early blastocyst stage. This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM. The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001). Blastocysts from MF1 x MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM. Blastocysts from C57BL6/CBA x C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM. When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 x MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005). MF1 x MF1 embryos and C57BL6/CBA x MF1 embryos of 60-79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA x C57BL6/CBA. When strain was compared MF1 x MF1 embryos of 60-79 cells had a significantly greater apoptotic index than C57BL6/CBA x MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA x C57BL6/CBA in M16 (P = 0.019). Our data suggest that genetic make-up and the chemical composition of simple medium are equally important in determining the level of apoptosis.     

MicroRNA expression and regulation in mouse uterus during embryo implantation

MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miR-NAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.     

Effects of leukaemia inhibitory factor on embryo implantation in the mouse

Leukaemia inhibitory factor (LIF) is a pleiotrophic cytokine. Recent reports indicate that LIF is relevant to murine embryo implantation. In this work, results of indirect immunofluorescence under a confocal microscope illustrated that LIF was mainly located in the uterine lumen and uterine epithelial cells in pregnant mice on day 4. The number of embryos implanted in pregnant mice on day 8 decreased significantly after injection of 3 microg LIF antibodies into a uterine horn (P<0.001), which demonstrated again that LIF is a critical factor for embryo implantation. In a co-culture system, LIF (0.1 ng/ml, 1 ng/ml, 10 ng/ml and 100 ng/ml) significantly enhanced the blastocyst outgrowth after 24, 48 or 72 h of co-culture, and outgrowth areas after 72 h of co-culture. Conversely, 5 microg/ml and 10 microg/ml, but not 1 microg/ml, LIF antibodies decreased the percentage of blastocysts with outgrowth; only 10 microg/ml LIF antibody inhibited blastocyst outgrowth area significantly (P<0.001). However, neither LIF nor its antibodies changed embryo attachment. Analysis of correlation showed that the effects of LIF or its antibodies on the blastocyst outgrowth were dose-dependent. In summary, different pathways may exist to regulate the blastocyst attachment and outgrowth on a monolayer of uterine epithelial cells. LIF protein from the maternal uterus exerts an essential role in embryo implantation in the mouse, which is mediated by stimulating trophoblast outgrowth, but not by promoting the attachment.     

The content of pyridine dinucleotides in the mouse embryo before implantation

Nicotinamide adenine dinucleotide (NAD) content was found to decrease in mouse embryos during cleavage and to increase again at the blastocyst stage. The first enzyme involved in biosynthesis of NAD from nicotinamide is nicotinamide mononucleotide (NMN)-pyrophosphorylase. No such enzymatic activity was found in the embryos, but NAD-glycohydrolase activity was relatively high 24–48 hours after conception. Enzyme activity decreased in the blastocyst. The results are relevant to understanding the regulation of metabolism in preimplantation embryos.     

Apoptosis in the developing zebrafish embryo.

Apoptosis is a major part of the normal development of many organ systems and tissues. The zebrafish (Danio rerio) has become a useful model for studying early development, and recent advances in techniques used to label apoptotic cells have made it possible to visualize apoptotic cells in this model system. We have used the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to describe the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 h postfertilization. By counting labeled apoptotic cells, we have demonstrated transient high rates of cell death in various structures during development, and we have correlated these peaks with previously described developmental changes in these structures. Our analysis has focused on the nervous system and associated sensory organs including the olfactory organ, retina, lens, cornea, otic vesicle, lateral line organs, and Rohon-Beard neurons. Apoptosis is also described in other non-neural structures such as the notochord, somites, muscle, tailbud, and fins.     

Inhibition of rat embryo implantation in the gossypol-treated uterine horn

We have developed a method to test the effect of gossypol on prevention of embryo implantation in the uterine horn. On the day of proestrus, gossypol (at a dose of 50, 100, 150, 200 and 500 mug per uterine horn was injected directly into the lumen of the right uterine horn. The left uterine horn was injected with 100 mul buffer. The rats were then mated with fertility proven males on the same day. The day of sperm-positive vaginal smear was designated as Day 0 of pregnancy. The number of implantation sites in both control and gossypol-treated horns was examined on Day 8 of pregnancy by laparotomy. The number of pups born was counted after parturition. At laparotomy, the percentages of pregnant animals with positive implantation sites in the gossypol-treated uterine horn (at a dose of 500, 200, 150, 100 and 50 mug per uterine horn) were 0, 0, 0, 10 and 44%, respectively. By contrast, implantation sites were present in 100% of the control horns of the same rats. The average numbers of total implantation sites in both horns vs the number of pups born to gossypol-treated animals using 500, 200, 150, 100, and 50 mug doses were 5.60 +/- 1.25 vs 4.00 +/- 1.00, 5.83 +/- 1.30 vs 4.70 +/- 1.10, 5.80 +/- 1.10 vs 5.50 +/- 1.20, 11.50 +/- 1.00 vs 9.50 +/- 1.50 and 11.67 +/- 1.20 vs 9.30 +/- 1.20, respectively. Gossypol metabolite completely inhibited embryo implantation when administered at 5.30 mug per uterine horn. The potency of the gossypol metabolite in preventing embryo implantation is estimated to be at least 28 times higher than the parent compound.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.     

血管生长抑素抑制小鼠胚胎植入的最低有效量及其作用机理

血管生长抑素是一种新的抑制血管生成的因子，它能够特异性地抑制血管生成。本文通过宫角注射法对血管生长抑素抑制小鼠胚胎植入的最低有效量进行了探讨，并通过明胶酶谱分析和免疫印迹等方法研究了不同剂量血管生长抑素对MMP-2和MMP-9活性及蛋白表达的影响。实验表明，宫角注射4μg血管生长抑素是抑制小鼠胚胎植入的最低有效剂量，这一最低有效剂量显著抑制了小鼠子宫MMP-2和MMP-9的活性及蛋白的表达[动物学报49(3)：332—338，2003]。     

Changes in secreted uterine proteins associated with embryo implantation in the mouse

A dual-label ratio method was used in conjunction with two-dimensional polyacrylamide gel electrophoresis to measure the relative changes in rates of production of individual secreted proteins by mouse uteri at the start of the process of decidualization. A characteristic pattern of differential changes in the rate of synthesis and secretion of the proteins was found to be associated with development of a positive Pontamine Blue reaction at the site of embryo implantation. These changes were compared with those associated with development of experimentally induced deciduomata and although the patterns were similar, presumably reflecting common processes in transformation of the endometrium, there was preferential enhancement of a subset of small (Mr 14,000-20,000) acidic proteins in the authentic implantation sites. It is suggested that this embryo-dependent modification of constitutive changes associated with decidualization reflects a form of embryo-maternal signal-response mechanism that may be important for the process of implantation in mice.