DNA synthesis in the mouse blastocyst during the beginning of delayed implantation

The spatiotemporal pattern of DNA synthesis in the mouse embryo at the beginning of metabolic dormancy was examined. Embryos were recovered from females at intervals following ovariectomy at 1100 hours on day 4 of pregnancy, incubated in vitro for 1 h in the presence of [3H]thymidine, and prepared for light microscopic autoradiography. The proportion of labeled cells in the embryo remained high (40-60%) for 18 h after ovariectomy and then declined gradually to 12% by 96 h. However, analysis of individual cell subpopulations showed that the decline was not uniform in all regions of the blastocyst. Labeling was high over the inner cell mass (ICM) during all time intervals in the study, while labeling over the mural trophoblast cells declined sharply by 24 h after ovariectomy. Labeling over the polar trophoblast also declined but had values that were intermediate between the ICM and mural trophoblast regions of the blastocyst. These regional differences in DNA synthesis during the arrest of development suggest that intermediate steps are involved in control of DNA synthesis in the embryo and that the ICM may play a role in the different responses of the trophoblast cell populations.     

Morphological changes in the blastocyst of the western spotted skunk during activation from delayed implantation

Blastocysts collected from the spotted skunk during delay of implantation, early activation and late activation demonstrate three-tiered growth and developmental changes. The slow-growing blastocyst from the several months of delay is small (less than 1.1 mm) with a rounded inner cell mass consisting of clusters of rounded, lipid-filled cells. During the several days of early activation, the lipid in both inner cell mass and trophoblast diminishes, polyribosomes increase in number, and the endodermal layer differentiates as the blastocyst grows (1.2-1.6 mm). At activation the inner cell mass flattens, becomes uncovered by polar trophoblast, and forms a disc of columnar epiblast cells. The blastocyst expands rapidly during the last 24-48 h prior to implantation to 1.7-2.0 mm, and the trophoblast becomes cuboidal with a marked endocytotic apparatus. The morphological evidence, together with previous studies of protein and RNA synthesis, suggests a tooling-up period during early activation with progressive increases in rates of growth and differentiation in the last hours as implantation approaches.     

Cell division and death in the mouse blastocyst before implantation

Summary The numbers of cells in the trophectoderm (TE) and inner cell mass (ICM) of mouse blastocysts were counted by differentially labelling their nuclei with two polynucleotide-specific fluorochromes. Blastocysts recovered from the uterus at intervals between their formation early on Day 4 to the initial stages of implantation on day 5 were analysed. TE cell number increase was initially rapid, indicating some synchronisation of the sixth division, but slowed down progressively and plateaued on Day 5, possibly due to the onset of primary giant cell formation. ICM cell number increase was slower than the corresponding TE cells. As a result, TE cell number more than quadrupled, whereas ICM cell number only doubled over this period. Although the mitotic index of both populations of cells fell steadily, there was no significant difference between them. The decline in the proportion of ICM cells, therefore, is likely to be due to cell death, first detected in early blastocysts and predominantly located in the ICM. In addition, however, a contribution of ICM cells to the overlying polar TE cannot be excluded.     

Cell surface changes on the mouse blastocyst at implantation

Cell surface changes on the trophectoderm of the mouse blastocyst have been followed in the periimplantation period using electronhistochemical techniques. Examination of the ability of the trophectoderm to bind positively charged colloidal iron particles before and after enzyme treatment has shown that sialic acid-containing glycoproteins make a considerable contribution to the negative charge on the blastocyst surface. At implantation these membrane components are lost or undergo modification independently of direct maternal influence as indicated by a marked decline in colloidal iron binding at this time, both in vivo and in vitro. The findings are discussed in relation to other surface changes on the blastocyst and to the initiation of implantation.     

Epithelial cell function during blastocyst implantation

In response to the ovarian secretion of progesterone and estrogen during early pregnancy, the mammalian uterus develops the capacity to perform complex cellular activities which occur before and after blastocyst implantation. Luminal epithelial cells participate in regulation of the metabolism of the blastocyst through the control of its humoral environment, provide an appropriate matrix for changes to occur at the interface between trophoblast and epithelium, and appear to transmit information from the blastocyst to the underlying stroma to initiate decidualization. With the completion of these functions during implantation in rodents, the epithelial cells self-destruct and are removed by phagocytic activity of the trophoblast. Control of both the endocytotic and secretory activity of luminal epithelial cells and their eventual self-destruction would require regulation of the Golgi-endoplasmic reticulum-lysosomes system within these cells. Progesterone secretion during early pseudo-pregnancy increases levels of cathepsin D, a lysosomal proteinase, in luminal epithelial cells by increasing the rate of enzyme synthesis. Progesterone pretreatment of ovariectomized rats followed by estradiol treatment results in the development of uterine sensitivity to deciduogenic stimuli. The number of proteins which are synthesized by luminal epithelial cells in response to estradiol to achieve this sensitivity has been determined. Epithelial cytosol proteins from rats treated with medroxyprogesterone acetate (3.5 mg sc) or medroxyprogesterone acetate plus estradiol (200 ng sc) were separated by two dimensional polyacrylamide gel electrophoresis. The synthesis of two proteins increased after 8 h of estradiol treatment and the synthesis of another three was increased by 12 h. The increased synthesis of these proteins could be related to changes in the capacity of the luminal epithelial cell for prostaglandin synthesis. The epithelial capacity for prostaglandin synthesis increases during pseudopregnancy to maximum levels at the time of maximum sensitivity to deciduogenic stimuli. Epithelial prostaglandin synthetic capacity may also depend upon the accumulation of prostaglandin precursors within these cells. Estradiol treatment of medroxyprogesterone acetate pretreated ovariectomized rats increased the arachidonic acid content and composition of epithelial phosphatidyl choline but the increases were not statistically significant. These changes in protein and lipid synthesis controlled by progesterone and estrogen would appear to contribute to the cellular activities of the luminal epithelium during early pregnancy.     

Changes in the 17 beta-hydroxysteroid dehydrogenase activity of mouse blastocysts during delayed implantation

The rate of estrone (E1)----estradiol-17 beta (E2) or E2----E1 conversion catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was determined for each mouse embryo in modified F-10 medium containing 0.95 microM 3H-E1 or 3H-E2. During delayed implantation, the E1----E2 conversion rate was decreased (p less than 0.005) from 5.69 +/- 0.34 fmol/h/blastocyst on Day 5 to 3.50 +/- 0.46 fmol/h/blastocyst on Day 9, whereas E2----E1 was increased (p less than 0.005) from 7.44 +/- 1.08 to 18.60 +/- 2.04 fmol/h/blastocyst. After estrogen injection, the Day 9 implanting blastocyst showed an increase (p less than 0.005) in E1----E2 conversion to 9.05 +/- 0.64 fmol/h/blastocyst but a slight, insignificant decrease in E2----E1 conversion to 14.2 +/- 1.82 fmol/h/blastocyst. A similar trend was also observed in Day 5 implanting blastocysts when compared to Day 5 delayed blastocysts. Thus, 17 beta-HSD activity in delayed blastocysts favors E2----E1 over E1----E2 conversion in a ratio of 5:1. After estrogen induction of implantation, the E1----E2 conversion rate is stimulated and the ratio of E2----E1 to E1----E2 rate is decreased to 1.5:1. The results suggest that 17 beta-HSD activity may be involved in blastocyst implantation.     

Persistence of embryonic RNA synthesis during facultative delayed implantation in the mouse.

The status of embryonic RNA synthesis during facultative delayed implantation in the mouse has been examined by radiolabeling in vitro and in utero, and by assay for endogenous RNA polymerase activity. Under conditions that do not activate delayed blastocysts in utero, embryos were shown to be able to transport and incorporate [³H]uridine into RNA as early as 5 min after intralumenal instillation of label on Day 5 of delay. Assay for endogenous RNA polymerase demonstrated functioning enzyme(s) in blastocysts on Day 5 of delayed implantation. Rates of incorporation of label in vitro under nonactivating conditions indicated a reduction, from normal Day 5 blastocyst levels, of 52% on Day 2 and 36% on Day 5 of delay. Relative rates of uptake of [³H]uridine by blastocysts on Day 5 of delay were reduced by approximately 60% from rates observed in predelay embryos on Day 5 of pregnancy. Estrogen-induced activation of embryos in utero was not associated with an increased relative rate of ³H]uridine uptake or incorporation during the first 24 hr following activation on Day 5 of delay. The findings demonstrate that RNA synthesis persists in the mouse blastocyst during delayed implantation, although at a somewhat reduced level. Implications of these results relevant to the maternal regulation of embryonic growth and implantation are discussed.     

Evaluation of mouse blastocyst implantation rate by morphology grading

The aim of our study is to observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse embryos obtained from the superovulated-ICR mice were cultured in vitro from 1-cell zygotes to blastocysts. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. They were independently transferred into the uterus of recipient females mated with vasectomized male mice on 96 hours after the zygotes were cultured in vitro. The successful implantation was checked by injection of Chicago Sky Blue 6B on the second day after embryo transfer. Although there was no significant difference in the implantation rates between the grade III and grade II, grade I was significantly decreased, as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE), as compared with grade III. These findings indicate that the expanded and hatching blastocyst selections for embryo transfer in in vitro fertilization were evaluated with the high implantation rate.     

p16INK4a在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨在检测肿瘤抑制基因p16INK4a(inhibitor of cyclin-dependent kinase 4a)在早孕小鼠子宫内膜中的表达规律,探讨p16INK4a在小鼠胚胎着床过程中的作用.采用荧光定量PCR(FQ-PCR)和免疫组织化学方法分别检测未孕小鼠及孕小鼠第2、3、4、5、7天子宫内膜p16INK4a mRNA和蛋白的表达;子宫角注射p16INK4a抗体观察胚泡着床数.FQ-PCR结果显示孕小鼠子宫内膜组织p16INK4amRNA的表达高于未孕小鼠,且随着妊娠天数的增加呈现表达逐渐增强的趋势,到妊娠第5天达到最高,后渐降.免疫组织化学分析显示p16INK4a蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射p16INK4a抗体后胚泡着床数明显减少.以上结果提示,P161INK4a在妊娠早期子宫内膜持续表达,可能参与胚泡着床.     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor α (TGF-α) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100–500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1–100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors. Mol. Reprod. Dev. 50:54–62, 1998. © 1998 Wiley-Liss, Inc.     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

Ultrastructure of the endometrial blood vessels during implantation of the rat blastocyst

The ultrastructure of the blood vessels of the endometrium was analysed during implantation of the blastocyst in rats, at the time of appearance of the Pontamine Blue Reaction. Vessels from implantation sites and from interimplantation sites were compared. In vessels from implantation sites the endothelial cells showed fenestrations covered by diaphragms. In addition, small interruptions (gaps) between the endothelial cells were observed. These features were present in vessels larger than 5 micrometers in diameter and more than 100 micrometers away from the uterine epithelium, both in the mesometrial and antimesometrial wall of the endometrium. Vessels from interimplantation sites displayed neither fenestrations nor interruptions. The endothelial cells of the implantation sites displayed morphological signs of metabolic activation. These consisted of increased numbers of polyribosomes, well developed Golgi complexes and prominent nucleoli. The fenestrations and gaps in the vessel wall were interpreted as constituting the morphological basis for the increase in vascular permeability and the consequent edema which characterize the Pontamine Blue Reaction.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.