Retinoic acid decreases the viability of mouse blastocysts in vitro

BACKGROUND: This study was designed to examine the cytotoxic effect of retinoic acid on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryo development in vitro. METHODS AND RESULTS: Mouse blastocysts were exposed for 24 h to doses of 0, 0.1 micromol/l and 10 micromol/l all-trans retinoic acid and observed for their capacity to implant and develop during the early postimplantation period in vitro. When retinoic acid-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to reach a later stage of embryo development. Compared with the findings for the control blastocysts, exposure to retinoic acid resulted in a significant reduction in the average number of total cells in blastocysts and the trophectoderm/inner cell mass lineage. The effect was associated with a significant increase in the frequency of cells identified as being engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and Annexin V techniques. CONCLUSIONS: This is the first evidence that retinoic acid induces cell death (apoptosis) and inhibits cell proliferation in mouse blastocysts. This results in the retardation of early postimplantation blastocyst development and subsequent blastocyst death.     

Fertilization and early embryology: Selection of viable mouse blastocysts prior to transfer using a metabolic criterion

The success rate of human in-vitro fertilization (IVF) remainslow, with only -10% of embryos transferred resulting in a termpregnancy. A major contributor to this embryonic loss Is poorembryo development in vitro. Such poor development can be attributedto both chromosomal and anatomic anomalies in oocytes afterovarian stimulation and to suboptimal embryo culture conditions.The low success rate of IVF is compounded by an inability toselect those embryos most likely to implant after transfer (viable).Currently morphology is used almost exclusively as the solecriterion to decide which embryos are replaced. This procedureis not only subjective but has a poor correlation with subsequentdevelopmental competence. Therefore, the development of techniquesto quantify embryo viability prior to transfer will significantlyincrease pregnancy rates. We report here that the non-invasiveassessment of glycolytic activity (percentage of glucose convertedto lactate) in individual mouse blastocysts prior to transfercan be used successfully to identify viable embryos. Blastocystswith a low glycolytic activity, close to that of in-vivo developedblastocysts, had a significantly higher viability than thosewith abnormally elevated levels of glycolysis. Using glycolyticactivity as a marker of viability resulted in a four fold increasein the pregnancy rate compared with embryos selected at randomfor transfer. We propose that the success of clinical IVF canbe increased significantly by employing quantitative tests forviability.     

Metabolism of polyunsaturated fatty acids by mouse peritoneal macrophages: The lipoxygenase metabolic pathway

When resident macrophages from mice are incubated with exogenous polyunsaturated fatty acids, they produce lipoxygenic metabolites. To delineate this metabolic chart we used high pressure liquid chromatography and gas chromatography prior to mass spectrometry-computer system. The lipoxygenic activity of these cells leads to many compounds. Among them we describe the monohydroxylated metabolites and vicinal hydroxyepoxyenes. In the mechanism of formation of the latter unstable cyclic precursors might occur as intermediates between hydroperoxides and them. Dihydroxy compounds could arise from hydrolysis of unstable epoxide precursor which could be the second substrate of the glutathione transferase system and could lead to thioaminolipids.     

Submicroscopic localization of glycogen in mouse blastocysts developed in vivo and in blastocysts developed in vitro from two-cell embryos

Ultrahistochemical method according to Thiéry (1967) was used to determine the occurrence and localization of glycogen in blastocysts developed in vivo and in blastocysts developed from 2-cell embryos of the mouse for 62 to 64 h in in vitro culture. The presence of glycogen was found in blastocysts of both experimental groups. Glycogen had a monoparticulate character, i.e. the form of beta-granules, localized above all in the ground cytoplasm of cells. Their size varied from 10 to 30 nm. In the blastocysts developed in the physiological uterine environment the glycogen content was relatively low, trophoblasts cells containing regularly a higher amount of glycogen particles than embryoblast cells. In the blastocysts developed in the culture medium in the presence of currently used energy sources the distribution and content of glycogen were clearly graded according to the cell types. Compared with the in vivo-blastocysts, an abnormally high amount of glycogen was observed in the cytoplasm of trophoblast cells, a medium amount in the prospective endoderm cells and the minimum amount in the prospective ectoderm cells. The authors are of the opinion that differences in the accumulation of glycogen and its occurrence in the individual cells are in connection with their position in the blastocyst and with their relation to the surrounding microenvironment. It can be judged from the findings of glycogen deposits inside autophagic vacuoles and multivesicular bodies as well as inside extracellular located sacs that simultaneously with glycogen accumulation there also proceeds its partial degradation in lysosomal structures of blastocyst cells.     

整合素αv、α4在小鼠胚泡植入中的作用

目的：探讨整合素αv、α4在小鼠胚泡植入中的作用。方法：在体外培养的人蜕膜细胞中分别加入整合素αv、α4抗体，与小鼠胚泡共培养，观察整合素αv、α4抗体对小鼠胚泡在体外培养的人蜕膜细胞上的粘附和铺展的影响。结果：整合素αv抗体组，胚泡的粘附受到暂时抑制，48h时大部分胚泡才粘附并开始铺展；整合素α4抗体组，胚泡的粘附率不受影响，但铺展率及滋养层细胞的扩展面积以剂量依赖的方式受到抑制。结论：整合素αv、α4能够调节胚泡在人蜕膜细胞上的植入，其中整合素αv可能与胚泡的粘附有关，而整合素α4可能与胚泡侵入和滋养层细胞的进一步生长相关。     

The production of mouse fetal-placental chimeras using trisomy 16 and euploid blastocysts

Summary By means of a combination of immunosurgery and a modified method of microsurgery, blastocysts were reconstructed to produce viable chimeric fetal-placental units. Reciprocal reconstituted blastocysts were produced using euploid and trisomy 16 blastocysts. Reconstructed blastocysts yielded significantly smaller fetuses at day 17 of pregnancy than simultaneously transferred control blastocysts (mean body weight 0.49 g vs 0.64 g, P<0.01). However, apart from reduced size, no abnormalities were observed for any euploid fetus-euploid placenta construct. The three reconstructed blastocysts that yielded a trisomie fetustrisomic placenta were viable when examined on day 17 and displayed the abnormalities typical of mouse trisomy 16. No reconstructed blastocyst that yielded a trisomic fetus-euploid placenta or a euploid fetus-trisomic placenta was viable beyond day 13 of development. One case in which a trisomie fetus had a placenta that was chimeric (euploid/trisomic) examined on day 17 displayed the abnormalities typical of a trisomie fetus but the placenta appeared histologically normal. The findings suggest that there is a coordination of the development of the fetus and the placenta that is essential for the development of the fetus.     

Rapid progression of passaged mouse hepatocarcinoma associated with the loss of cell polarity

Two transplantable differentiated mouse hepatocarcinomas were obtained in B6 D2 F1 mice from primary tumors induced by initiation (NDEA)-promotion (phenobarbital) protocol. Both HC were slowly growing with time from passage to passage of 5-7 months (s-HC). A fast-growing variant with time between passages of 2-3 weeks appeared in one mouse on the third passage (f-HC). The three HC strains constantly retained their phenotype. The s-HCs were characterized by prominent cell polarity according to organization of the cytoskeleton and distribution of domain-specific membrane-associated markers. Cell polarity was completely destroyed in the fast-growing variant. Cell adhesion in the latter tumor was very low. An in vitro growing strain of f-HC was easily obtained enabling experimental study of regulation of progression in HCs.     

Observations on the development of mouse blastocysts transferred to the testis and kidney

The development of mouse blastocysts transferred to the kidney and testis was studied by light and electron microscopy. Approximately 50% of the blastocysts transferred to both sites survived for more than five days. Although trophoblast proliferation was characteristic of all 31 successful transplants which were serially sectioned, inner cell masses differentiated in only nine. Transplants were invasive in kidney but were encapsulated in testis. In both organs, however, transplants produced considerable hemorrhage. That trophoblast invades by phagocytosis was not substantiated in this study nor was there evidence that trophoblast elaborates a cytolytic factor. Although transplants caused gross lesions in the host organs, the cytology of cells in close proximity to invading trophoblast was almost normal. The only abnormal features observed were the presence of fine fibrillar material in the basal cytoplasm of renal tubule cells and a thickening of their basement membranes. No correlation could be demonstrated between the antigenic dissimilarity of host and transplant and extent of host tissue response.     

The rate of development and time of transfer play different roles in influencing the viability of human blastocysts

Improved embryo culture protocols now render more feasible the possibility of obtaining human blastocysts after in-vitro fertilization. In this study we present: (i) results of blastocyst development from supernumerary embryos after co-culture on green monkey kidney epithelial cells and (ii) pregnancy rates after transfer of frozen blastocysts. In addition, we have examined the influence of the day of blastocyst freezing and the day of transfer after the luteinizing hormone (LH) peak on pregnancy and implantation rates. Of 423 supernumerary embryos, 200 developed to the blastocyst stage (47.3%). By days 5 and 6, 67% of the blastocysts had reached the blastocyst stage, and were frozen, compared to 28.5% by day 7. When we compared the cases where only blastocysts frozen on days 5 and 6 were transferred compared to those frozen and transferred on or after day 7 the pregnancy rates were 7/18 (38.9%) and 1/16 (6.2%) respectively. In contrast, when we examined the influence of the day of transfer we found that pregnancies were established from day 5 up to day 9 post LH peak. Based on these results, we suggest that every attempt should be made to increase the development rate of supernumerary embryos to the blastocyst stage, as it appears that the quality of blastocysts transferred, as shown in this study by rate of development, plays a more crucial role than the timing of transfer.      

Transmission electron microscopy of mouse blastocysts activated and growth-arrested in vivo and in vitro

Summary Blastocysts from mice in a state of delayed implantation were examined by electron microscopy, either directly or after a culture period, to find out if activation and growth arrest in vitro were similar to the corresponding stages in vivo. It was found that activation in vitro, obtained by culturing the blastocysts in an outgrowth medium, was accompained by morphological signs similar to those during oestrogen activation in vivo, namely an increase in polyribosomes and glycogen granules. Analogously, growth arrest in vitro, obtained by culturing the blastocysts in a medium deprived of glucose, arginine and leucine, was accompanied by a morphology similar to that of blastocysts obtained during delay of implantation, namely scattered ribosomes of the monosome type, few membranes of endoplasmic reticulum and a lack of glycogen granules, all signs of a low metabolism. However, the morphological parallelism between the in vitro and the in vivo systems does not necessarily mean that the growth-controlling factors are the same in both cases.Activated blastocysts were transplanted to uteri of mice in delay of implantation to characterize the trophoblast ultrastructure four days after the transplantation. The experiments demonstrated that both in blastocysts activated for 24h in vivo and in those activated for 6 h in vitro the morphology reverted to that of inactive blastocysts, indicating that the functional inactivity is related to reversion into a morphologically inactive state rather than to uterine blocking of a morphologically active state.     

Use of urinary taurine and creatine as biomarkers of organ dysfunction and metabolic perturbations

We have shown that urinary taurine and creatine may be used as biomarkers of pathological and biochemical lesions. Detection of changes in the urinary concentration of these two low-molecular-weight metabolites indicates biochemical lesions which may also be associated with pathological damage. Thus changes in protein or glutathione metabolism will lead to changes in urinary taurine. Hepatotoxicants which cause necrosis, but do not affect glutathione status or reduce bile flow, cause taurinuria and creatinuria. Hepatotoxicants which cause fatty liver only cause taurinuria. Testicular toxicants only cause creatinuria. Compounds which result in increased glutathione synthesis, stimulate protein synthesis or reduce bile flow decrease urinary taurine. A combination of the two markers, therefore, can be used to help diagnose a variety of biochemical and pathological lesions.Originally presented at ECCP 95.     

Stereological study of mouse uterine and in vitro grown blastocysts: Cell numbers and volumes

Mouse blastocysts were studied to determine if there were differences in cell number and volumes between those that were (1) derived from the uterus prior to implantation on the afternoon of day 4 of pregnancy and (2) those that were cultured for 72 hr from two-cell-stage embryos. Blastocysts were fixed, embedded in resin, and serially sectioned at 1.5 or 2 μm. Photographic prints of alternate sections were used to count the numbers of inner cell mass (ICM) and trophectoderm cells. Cavalieri's direct estimator was applied to the same prints to estimate the volume of the whole blastocyst. Point counting was used to determine the volumes of the ICM, trophectoderm, and zona pellucida. The number of cells and size of the ICM were similar between the two groups of blastocysts, although it was found that the ICM of uterine embryos that did not have a zona pellucida were smaller than the ICM of those that did. There were twice as many trophectoderm cells in the blastocysts that were cultured from two-cell embryos, and these cells were also found to be larger. Furthermore, the volume of the zona pellucida was less in the uterine blastocysts. This study indicates that, while trophectoderm proliferation is enhanced in vitro, the ICM is more constant and thus may be self-regulating and independent of the growth conditions of the blastocyst as a whole. This study also suggests partial zona lysis occurs in utero and occurs either at a reduced rate or not at all in vitro. © 1993 Wiley-Liss, Inc.     

Identification of genetic loci associated with paralysis, inflammation and weight loss in mouse experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is an inducible inflammatory and demyelinating disease of the central nervous system (CNS). Susceptibility to this disease is heritable and is demonstrated by the development of an ascending paralysis accompanied by a loss in body wt 2-3 weeks following immunization with proteins derived from CNS myelin. In a previous genetic analysis of susceptibility to EAE in a cross between susceptible SJL/J mice and resistant B10.S mice, we found suggestive evidence of linkage with disease susceptibility at the telomeric end of chromosome 2 and in the central region of chromosome 3. To define these associations more precisely and to investigate the genetic factors controlling measurable phenotypes of EAE, we performed a new analysis with a larger number of mice. The results now indicate that the chromosome 2 locus significantly influences EAE-related weight loss (P = 6.7 x 10(-5)) and that the chromosome 3 locus is linked with the development of paralysis. In addition, an intriguing inheritance pattern was revealed in which female backcross mice generated from B10.S female x (B10.S x SJL/J)F(1) male parents experienced significantly more EAE-related weight loss (P = 1.2 x 10(-4)) than females generated from F1 female x B10.S male parents. After controlling for this inheritance, a new locus at the centromeric end of chromosome 8 was identified that significantly influences both the development of paralysis (P = 8.2 x 10(-6)) and the incidence of CNS inflammation (P = 7.0 x 10(-5)) in EAE.     

小鼠囊胚细胞凋亡和半胱氨酸天冬氨酸蛋白酶3表达与维甲酸的诱导作用

背景：细胞凋亡是维甲酸导致早期胚胎异常发育的原因之一,而半胱氨酸天冬氨酸蛋白酶3介导的细胞凋亡是细胞凋亡的主要途径。 目的：观察维甲酸对体外培养小鼠囊胚细胞凋亡的影响,以及对囊胚细胞中半胱氨酸天冬氨酸蛋白酶3表达的改变。 方法：获取妊娠3.5 d小鼠囊胚,分别在含0,1和10 μmol/L维甲酸的M199培养基中培养24 h,用带有荧光标记的原位末端标记法观察维甲酸对小鼠囊胚细胞凋亡的效应;并用免疫组织化学S-P法检测维甲酸对小鼠囊胚半胱氨酸天冬氨酸蛋白酶3的表达。 结果与结论：所有组均观察到囊胚细胞凋亡和半胱氨酸天冬氨酸蛋白酶3表达,高剂量维甲酸组中囊胚细胞凋亡荧光值,半胱氨酸天冬氨酸蛋白酶3表达的平均吸光度值和阳性面积率均显著升高(P < 0.01)。结果提示维甲酸可促进体外培养小鼠囊胚细胞凋亡和及半胱氨酸天冬氨酸蛋白酶3表达,证实了维甲酸对小鼠胚胎的发育的细胞毒性作用。     

Unsaturated fatty acids and viability of Helicobacter (Campylobacter) pylori.

Helicobacter (Campylobacter) pylori was found to be sensitive to the toxic effects of an unsaturated fatty acid (arachidonic acid). Data are presented that support the hypothesis that exogenous catalase added to basal media enhances the growth of H. pylori by preventing the formation of toxic peroxidation products from long-chain unsaturated fatty acids.     

A comparative study of attachment of human, bovine and mouse blastocysts to uterine epithelial monolayer

The in-vitro attachment of human, bovine and murine blastocyststo monolayer cultures of uterine epithelium were studied bytransmission electron microscopy. The human trophoblastic cellsintrude between uterine epithelial cells forming a multilayerduring attachment in vitro, thus resembling the intrusive typeof penetration observed in vivo. The bovine trophoblastic outgrowthresembled an epithelio-chorial attachment as the trophoblastformed an attachment plate on top of the endometrial cells withoutpenetration. In the murine attachment study, the trophoblastcells immediately displaced the uterine cells and formed contactwith the culture vessel.     

Amino acids 1 to 422 of the spike protein of SARS associated coronavirus are required for induction of cyclooxygenase-2

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV). To evaluate the molecular mechanisms involved in the viral infection, in this study, we investigated the role of SARS-CoV Spike (S) protein in the regulation of cyclooxygenase-2 (COX-2). Expression of COX-2 stimulated by the S protein was verified by RT-PCR and western blot assay. To explore the relationship between S and COX-2, we constructed a series of plasmids containing truncated N-terminal fragments of the SARS-CoV S gene (designated from Sa to Si), which encoded truncated S proteins, and investigated whether these truncated proteins could induce effective expression of COX-2 in 293T cells. Our results showed that S_d that encoded a truncated S protein with 422 amino acid residues (from 1 to 422 aa), a part of 672 amino-acid S1 subunit is crucial for the induction of COX-2 expression. Immunofluorescence examinations also give the evidence that these N terminal 422 amino acids of the S protein were also required for the correct localization of the protein. We also compared S protein sequences of SARS-CoV isolated during the SARS break with that from palm civets, a possible source of SARS-CoV found in humans. S protein residues (344, 360), which mutated in the epitome from palm civet to human being were characterized in 3D modeling of 252–375 amino acid fragment. Collectively, these results indicate that S protein of SARS-CoV induces the expression of COX-2 and an N-terminal fragment of the Spike protein is crucial for the induction. Our finding may provide clue for the induction of inflammation by SARS-CoV and cast insight into the severity of the SARS epidemic.