Fertilization and early embryology: Differential effect of epithelial cell-conditioned medium fractions on preimplantation mouse embryo development

Coculture studies using preimplantation embryos have led toa number of conflicting studies. In the human, ethical considerationshave led to the preferential use of epithelial cell lines asdistinct from human Fallopian tube cells. In an attempt to isolatefactors influencing embryo development we have cultured 2-cellOF1 mouse embryos in media [Ménézo's B2 and Whittingham'sT6 supplemented with vitamins and amino acids (T6VA)] conditionedon two types of kidney epithelial cells (MBDK and Vero). Differentmolecular weight fractions of conditioned medium were used toshow the absence or presence of specific embryotrophic factors.With MDBK cells, B2 conditioned medium enhanced embryo developmentup to the blastocyst stage, while no blastocysts developed inB2 alone. When using T6VA medium, both the control and conditionedmedia showed a high percentage of blastocyst formation (57.0and 54.0% respectively), while the different molecular weightfractions showed no added improvement. With Vero cells, B2 alone,B2 conditioned medium and fractions were all detrimental toembryo development. A high percentage of blastocyst formation(between 64.7 and 75.8%) was observed in T6VA alone, T6VA conditionedmedium and fractions. Low blastocyst formation in a controlmedium can show strong positive results when medium is conditionedby cells. In contrast, a good base medium, such as T6VA, canequal the results using conditioned medium. Different cellsin contact with different types of medium show variability inthe pattern of responses, highlighting the presence of falsepositives in coculture studies.     

Chromosomally abnormal cells are not selected for the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage

BACKGROUND: Although well defined for embryos at cleavage stages, the occurrence and frequency of chromosomal aberrations in human blastocysts is relatively unknown. It has been reported that only one in four blastocysts is comprised totally of chromosomally normal cells. One of the selection mechanisms for the embryo proper to become free of these chromosomally abnormal cells would be to sequester them to the extra-embryonic compartment during development. The study aim was to investigate whether such a mechanism of selection exists in human preimplantation embryos. METHODS: Inner cell mass (ICM)/trophectoderm (TE) differentiation was performed, followed by fluorescence in-situ hybridization (FISH), to study the chromosomal distribution in both populations of cells. RESULTS: Of the 94 successfully analysed blastocysts, 68.8 +/- 1.5% of all analysable nuclei per blastocyst showed a disomic chromosomal content. Only 22.6% of blastocysts analysed were classified as normal. Of the embryos classified as abnormal at the blastocyst stage, 11.9% showed a simple mosaic pattern and 32.1% a complex mosaic pattern. An equally large group of blastocysts showed either a chaotic pattern (16.7%), or the chromosomal pattern could not be classified. The average degree of normal cells in the ICM (67.9%) was similar to the degree observed in the TE (69.5%). CONCLUSIONS: These findings indicate that chromosomally abnormal cells are not preferentially segregating to the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage. Hence, other mechanisms should be responsible for an absence of chromosomally abnormal cells in the embryo proper at later stages of development. One possible mechanism might be the elimination of the chromosomally abnormal cells by selective cell death activation.     

Parvalbumin and calbindin are differentially distributed within primary and secondary subregions of the mouse auditory forebrain

The calcium binding proteins parvalbumin and calbindin are thought to differentially regulate physiological functions and often show complementary distributions in the CNS. Our goal was to determine parvalbumin and calbindin distributions in the different subdivisions of mouse auditory thalamus and auditory cortex. Following fixation, FVB mouse brains (postnatal days 38-80) were sectioned along coronal and horizontal planes, then processed for parvalbumin and calbindin immunohistochemistry (antibodies: parvalbumin pa-235, calbindin-d-28k cl-300). Strong complementary differences in calcium binding protein distributions were found in mouse auditory thalamus. The ventral division of the medial geniculate, which is the principal relay to primary auditory cortex, exhibited dense parvalbumin but weak calbindin immunoreactivity. In contrast, most of the 'secondary' auditory thalamic regions surrounding the ventral division showed strong calbindin and lighter parvalbumin levels. Thus, the mouse auditory thalamus is composed of a parvalbumin positive 'core' surrounded by a calbindin positive 'shell'. Parvalbumin immunoreactivity was also more prominent in the primary auditory cortex than in the secondary belt auditory cortex. Calbindin immunoreactivity in auditory cortex was less clearly divided along primary/secondary lines, especially in supragranular layers. However, within infragranular layers, there was heavier staining in belt areas than in primary auditory cortex. In auditory thalamus, parvalbumin labeling was largely confined to the neuropil, whereas calbindin labeling involved somata and neuropil. In auditory cortex, somata and neuropil were positive for both proteins.In summary, the calcium binding proteins parvalbumin and calbindin were found to be differentially distributed within the primary and non-primary regions of mouse auditory forebrain. These differences in protein distribution may contribute to the distinct types of physiological responses that occur in the primary vs. non-primary areas.     

Fertilization and early embryology: Granulosa cells improve human embryo development in vitro

A total of 17 couples with repetitive implantation failure aftertransfer of fresh or frozen—thawed embryos had half oftheir zygotes cultured in standard conditions and frozen atday 2 after insemination, and the other half cocultured withautologous granulosa cells and transferred at the morula orblastocyst stage at day 5 or 6 after oocyte retrieval. At theend of the culture period, supernatants of cocultures were recoveredfor steroid assays. Monolayers were stained for granulosa cellgrowth and morphological assessment. We observed that granulosacells improve embryo development in vitro since 32 out of 60(53%) reached the morula stage and 18 (30%) the blastocyst stage,leading to a total of 83% embryos available for transfer (comparedwith 3% without coculture). The ongoing pregnancy rate of thesepatients who were selected because they had at least three previousimplantation failures, is only 5.9%, however, which is similarto the control group without coculture (6.3%). To conclude,granulosa cells improve embryo development but not the pregnancyrate after transfer of cocultured embryos in patients with multipleprevious implantation failures.     

Fas、FasL和Caspase-3在人胚早期脊髓发育中的分布

目的观察Fas、FasL和Caspase-3在人胚早期脊髓发育的表达。方法收集早期人胚35天,52天和60天标本9例,用4%多聚甲醛固定后制作8μm厚的石蜡切片。间隔取片分组,分别用Fas(效价1:250,Santa Cruz)、FasL(效价1:250,Santa Cruz)、Caspase-3(效价1:500,Sigma)抗体对5例人胚标本的10张切片行免疫组织化学ABC法染色。观察Fas、FasL和Caspase-3在人胚早期脊髓发育中的分布以及亚细胞定位。DAB棕色反应呈色。结果Fas免疫反应阳性的神经元主要分布在室管膜层、中间层和边缘层,可见胞浆和细胞核内染色。FasL免疫反应阳性产物主要分布在室管膜层和套层,可见细胞膜、胞浆和细胞核染色。Caspase-3免疫反应阳性产物主要分布在神经管的室管膜层和中间层,可见细胞核染色。结论在人胚发育早期,Fas、FasL和Caspase-3分布于脊髓,提示这些因子参与人胚早期脊髓的发育,参与神经元凋亡信号的传递。     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Fertilization and early embryology: Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos

In-vitro culture of 1-cell mouse embryos was used to assessthe influence of pentoxifylline on early embryonic development.If cultured in concentrations of 5, 10 or 50 µM, earlyembryonic development was unaffected and no differences in cellnumbers were noted in embryos reaching the blastocyst stage.However, at 3.6 and 7.2 mM, pentoxifylline inhibited cleavagefrom the 2-cell stage onwards. If 1-cell mouse embryos wereexposed for only 30 min to these concentrations, blastocystformation was found to be morphologically normal. However, cellnumbers of such blastocysts were significantly decreased afterexposure to pentoxifylline. These results may indicate thatexposure of gametes or zygotes to pentoxifylline should be avoidedas much as possible when this drug is used in human assistedreproduction. If administered at regular therapeutic doses,it is probable that no adverse effect on early embryonic developmentin vivo will occur. Further research is needed to confirm andelucidate the above findings.     

Effects of SAPK/JNK inhibitors on preimplantation mouse embryo development are influenced greatly by the amount of stress induced by the media

Stress-activated protein kinase/c-Jun kinase (SAPK/JNK) is thought to be necessary for preimplantation embryonic development (Maekawa et al., 2005). However, media increases SAPK/JNK phosphorylation and these levels negatively correlate with embryonic development (Wang et al., 2005). Culture-induced stress could confuse analysis of the role of SAPK in development. In this study, we tested how SAPK/JNK inhibitors influence embryonic development in optimal and non-optimal media and define the contribution of cell survival and proliferation to the embryonic response to these media. SAPK/JNK inhibitors retard embryonic development in suboptimal Ham's F10, but improve development in optimal potassium (K+) simplex optimized media (KSOM) +AA. In KSOM + amino acids (KSOM+AA), two SAPK/JNK inhibitors increase the rate of cavitation and hatching. These data suggest that (i) SAPK/JNK mediates the response to culture stress, not normal preimplantation embryonic development and (ii) SAPK/JNK inhibitors may be useful in ameliorating embryo stress caused by culture. To define the effects of media, we assayed the contribution of cell survival and proliferation and the differences in total cell number of cultured embryos. Embryos cultured from E3.5+24 h in the suboptimal medium (Ham's F10) induced significant but small increases in TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) positive cells. Bromodeoxyuridine (BrdU) incorporation in suboptimal Ham's F10 was significantly lower than in optimal KSOM+AA, suggesting that cell cycle arrest also contributes to slower increase in cell number in stressful media. This is the first report where TUNEL and BrdU were both assayed to define the relative contribution of cell cycle/S phase commitment and apoptosis to lessened cell number increase during embryo culture.     

Preimplantation embryology: Complement-binding proteins are strongly expressed by human preimplantation blastocysts and cumulus cells as well as gametes

Human preimplantation embryos, gametes and cumulus cells werestudied for expression of the complement-binding proteins CD46(membrane cofactor protein), CD55 (decay accelerating factor)and CD59 (membrane attack complex inhibitory factor) as wellas complement receptors type 1 (CRI), type 2 (CR2) and type3 (CR3). Both the CD55 and CD59 glycosyl phosphatidylinositol(GPI)-anchored proteins were expressed by the plasma membraneand zona pellucida of oocytes, early embryos and expanded preimplantationblastocysts; in contrast, CD46 was expressed only on the plasmamembrane. Cumulus cells consistently expressed CD46 and, moststrongly, CD59 whereas CD55 expression was variable. Monoclonalantibodies (mAbs) to CRI, CR2 and CR3 epitopes gave only occasionalreactivity on oocytes and were unreactive with blastocysts,spermatozoa and cumulus cells. CD46 is expressed only on thespermatozoal inner acrosomal membrane, and CD59 on the plasmamembrane; CD55 expression was confirmed on the plasma membraneas well as the inner acrosomal membrane. Control mAbs specificfor factor H were usually unreactive with gametes, blastocystsand cumulus cells. These data support the concept that gametesand early embryonic cells are protected from complement-mediatedattack by expression of CD46, CD55 and CD59, although thesecomplement-binding proteins may have additional roles in reproductiveevents. blastocysts/CD46(membrane cofactor protein)/CD55(decay accelerating factor)/CD59/cumulus cells     

Enhancement of mouse sperm motility by the PI3-kinase inhibitor LY294002 does not result in toxic effects on preimplantation embryo development

BACKGROUND: A reduced number of progressively motile sperm (as may occur in cases of asthenozoospermia or when cryopreserved spermatozoa are used for fertilization) limits the possibility of applying various assisted reproductive techniques (ARTs). We previously showed that incubation of sperm with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 increases sperm progressive motility and enhances the number of sperm recovered by capacitation protocols used in ART. METHODS AND RESULTS: In the present study, we investigate the motility-enhancing effects of this compound in epididymal mouse sperm, and examine the use of the mouse system to investigate the effect of LY294002 on oocyte fertilization and preimplantation embryo development. Our results show that neither pre-incubation of mouse spermatozoa with the inhibitor during in vitro capacitation nor the direct addition of LY294002 to the sperm-oocyte mixture significantly affects the process of fertilization and preimplantation development of embryos produced even when they developed in the presence of LY294002. CONCLUSIONS: The present data encourage the design of new drugs based on the molecular structure of LY294002, which may open up new options for the in vitro treatment of human/animal asthenozoospermia.     

Integrins and adhesion mlecules: Cell adhesion molecules on the oocyte and preimplantation human embryo

The presence of cell adhesion molecules on human oocytes, earlyembryos, and pre-hatched blastocysts was examined by indirectimmunofluorescence and compared to the distribution found onfirst trimester villous placenta with the same antibodies. Sixintegrin subunits (3, V, 1, 3, 4, 5) were observed consistentlythroughout preimplantation development. Evidence was also obtainedfor the presence of integrin subunits 2, 4, L, 2, and 7 on asmall number of oocytes. A more restricted developmental analysisof E-cadherin, ICAM-1, NCAM, and VCAM-1 demonstrated that thesecell adhesion molecules are also present on oocytes and earlyembryos. L-selectin was detected on oocytes but was not foundon 8-cell embryos. The oocyte and early blastomeres have complexsurfaces in which the integrin and CAM families are represented.     

神经营养素3和受体在早期人胚脑发育中的定位表达

目的:了解在早期人胚脑的发育过程中,大脑神经元的增殖情况;了解神经营养素3(NT-3)和受体TrkC在人胚脑发育过程中的影响。方法:用6周龄的人胚进行免疫组织化学ABC法染色,以PCNA作为大脑神经元的增殖的指标,NT-3和受体作为了解影响人胚脑发育因素的指标。结果:在人胚胎早期发育阶段,PCNA免疫阳性反应物主要分布于人胚前脑的神经细胞核中,NT-3免疫阳性反应物主要分布于前脑脑室神经上皮层的胞质中和神经细胞的轴突中,其受体TrkC散在分布于人胚前脑神经细胞膜;发育过程中NT-3及其受体在脑室神经上皮层上呈现不同程度的免疫阳性反应。结论:NT-3及其受体TrkC在早期的人胚胎脑发育中呈现表达,提示NT-3可能诱导神经细胞增殖,并协同其它生长因子促进轴突的生长,保证神经元胞体的存活。     

Insulin-like growth factor-binding proteins produced by Vero cells, human oviductal cells and human endometrial cells, and the role of insulin-like growth factor-binding protein-3 in mouse embryo co-culture systems

Co-culturing embryos on helper cells can mimic the in-vivo environment,thereby enhancing embryo development in vitro. Insulin-likegrowth factors (IGF) and their binding proteins (IGFBP) alsoenhance embryo development To investigate the kinds of IGFBPproduced by various cell monolayers and the effects of IGFBP-3on mouse embryo co-culture systems, 2-cell ICR mouse embryoswere cultured in either human tubal fluid medium alone or inthe presence of Vero cells, human oviductal cells or endometrialcells. The helper cells were analysed immunohisto-chemicallyto investigate the types of IGFBP produced by various cell monolayers.The concentrations of IGF-I and IGFBP-3 in media obtained fromthe culture of embryos alone, cells alone or cells plus embryoswere determined by radioimmunoassays. On day 7, more blastocystshatched in the co-culture groups (73% in the Vero cell group,76% in the endometrial cell group and 74% in the oviductal cellgroup) than in the control group (43%) (P < 0.0001). Theresults of immunohistochemistry revealed that (i) all threecell groups produced a lot of IGFBP-1, -2 and -3, but only alittle of IGFBP-4 and -5; and (ii) IGFBP-1, -2 and -3 were presentin blastocysts in either the presence or absence of helper cells.The IGF-I secreted by cell monolayers or embryos was undetectable(detection limit 0.83 ug/1). The IGFBP-3 concentrations in mediaobtained from co-cultured embryos and cells were significantlyhigher than in media without embryos (median values in oviductalcell culture medium, 165 versus 127 µg/1, P = 0.04; medianvalues in endometrial cell culture medium, 277.5 versus 183.5µg/1, P = 0.0002; median values in Vero cell culture medium,219 versus 120 µg/1, P = 0.011). Although IGFBP-3 concentrationin the medium that contained embryos alone was undetectableby radioimmuno-assay (detection limit 1.1 µg/1), immunohistochemistrydemonstrated the presence of IGFBP-3 in the embryos. Co-culturein systems in which there was an increased production of IGFBP-3led to an improved development of mouse embryos. IGFBP can improvethe binding of IGF to cell surface receptors of target tissue,and thus enhance the effect of limited IGF concentrations inpromoting embryo development in a co-culture system. We concludethat Vero cells, human endometrial cells and oviductal cellsproduce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a rolein embryotrophic potential by either regulating the action ofIGF or directly enhancing embryo development     

Complement regulatory proteins in early human fetal life: CD59, membrane co-factor protein (MCP) and decay-accelerating factor (DAF) are differentially expressed in the developing liver.

The human fetus appears to be capable of protecting itself from maternal complement (C) from an early stage in development by expressing the C regulatory proteins decay-accelerating factor (DAF), membrane co-factor protein (MCP) and CD59 on fetally derived trophoblast at the feto-maternal interface. In this study we have examined the ontogeny of these proteins within the fetus itself and have focused on the liver which represents a major site of haemopoiesis during development. Immunostaining revealed that DAF, MCP and CD59 are all expressed from at least 6 weeks of gestation in the liver but that these proteins display distinct distribution patterns. CD59 was broadly distributed both within the epithelial and haemopoietic compartments, but expression of C3 convertase regulators was more restricted. DAF expression was limited to isolated cells within haemopoietic nests and the epithelium was DAF-negative. Although MCP expression on haemopoietic cells was also limited, by contrast with DAF the developing hepatic epithelium was strongly MCP-positive. Typical CD59 and MCP components were observed in fetal liver extracts by immunoblotting, although liver MCP components consistently migrated 4000-5000 MW ahead of those observed on placental trophoblast. Differences in the distribution of these proteins were also observed between the fetal and adult liver. In particular, by comparison with fetal hepatic epithelium, there was an apparent loss of MCP expression from adult hepatocytes. Thus, MCP appears to be developmentally regulated in the human liver and is expressed in the absence of DAF on the early hepatic epithelium. Overall, this study suggests that C regulatory proteins, and in particular CD59 and MCP, are required from the very early stages of gestation within the fetus itself.     

Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model

BACKGROUND: The aim of this study is to model the processes of early embryopathy seen in human pregnancy complicated by maternal hyperglycemia secondary to maternal diabetes using a mouse embryo culture system. METHODS: Female mice were superovulated and mated in pairs. Two-cell embryos were harvested from the oviducts and cultured in vitro in KSOM medium (synthetic oviductal medium enriched with potassium) supplemented with 0.2, 5.56, 15.56 or 25.56 mM d-glucose. Cell proliferation, differentiation and apoptosis were assessed. Experiments were performed in constant, embryos exposed to a particular concentration of glucose (0.2, 5.56, 15.56 or 25.56 mM) from harvest to either Day 5 post fertilization (pf) or Day 8 pf, and fluctuating, embryos exposed to alternate high 25.56 mM and normal 5.56 mM concentrations of glucose between harvest and Day 5 pf, glycemic culture. RESULTS: Expected levels of blastocyst formation and hatching were seen at 0.2 and 5.56 mM concentrations of glucose but both were impaired at higher concentrations (chi(2), P < 0.005; P < 0.001). Total cell numbers (P < 0.002) and cell allocation to the inner cell mass (P < 0.01) were reduced, but with no evidence of enhanced apoptosis in the hyperglycemic cultures. Variation in hyperglycemic exposure of the embryos on Days 2, 3 and 4 showed no adverse effects of hyperglycemia up to 24 h, but 48 and 72 h exposures were equally embryopathic (P < 0.01). CONCLUSIONS: Hyperglycemic exposure for >24 h is toxic to early embryo development. These findings may explain the lower than expected implantation rates and higher than expected rates of congenital abnormality and early pregnancy loss seen in patients with diabetes, particularly those with poor diabetic control.