IGF-Ⅱ and IGFBP-1 reversely regulate blastocyst implantation in mouse

Insulin-like growth factor (IGF)-Ⅱ and IGF binding protein (IGFBP)-1, members of IGF family, are important in the cyclic development of endometrium and the blastocyst implantation. In the present study, the indirect immunofluorescence showed that IGF-Ⅱ and IGFBP-1 were specifically expressed at the maternal-fetal interface. In a co-culture system, IGF-Ⅱ significantly enhanced the attachment and outgrowth of the blastocyst on monolayer of uterine epithelial cells, while IGFBP-1 did not affect the blastocyst attachment, but markedly inhibited the blastocyst outgrowth. The results of zymography showed that IGF-Ⅱ enhanced the activities of MMP-2 and MMP-9, while IGFBP-1 did not affect the activities of MMP-2 and MMP-9. In conclusion, the equilibrium between the invasion of trophoblast and the inhibition of deciduas may be regulated by the interaction between the IGF-Ⅱ-expressing invading cytotrophoblast and maternal deciduas-derived IGFBP-1.     

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

Embryo implantation: A time for recalling and forwarding

The success of embryo implantation is a critical step towards further embryo development and pregnancy outcome. The observations and investigations on embryo implantation have been over a century. A huge body of knowledge has been accumulated in anatomy, histology, ultrastructure and hormonal regulation; as well as recently in depth information about molecular signaling pathways got from studies of genomic wide gene screening and specific gene deletion. The knowledge from basic research has also substantially helped to initiate and improve the Artificial Reproductive Technology （ART） in clinical applications. Now we＇ve known that the normal embryo implantation involves the embryo＇s development into an implantation-competent blastocyst and the synchronized transformation of uteri into a receptive stage. The interdependent relationship between the blastocyst and uterus involves complicated hormonal regulation and local paracrine, juxtacrine interactions. In this paper, we review some important historical findings regarding uterine receptivity and blastocyst activation, as well as some less discussed topics such as embryo spacing, embryo orientation. Further understandings on detailed mechanisms during the process of embryo implantation will help cure women infertility as well as develop new generation of non-steroids contraceptives.     

Expression, distribution and function of the focal adhesion kinase (pp125FAK) during murine ectoplacental cone outgrowthin vitro

Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

Expression of LIF,VEGF, CD57 and CD68 after the transfer of rat embryos to mouse uteri

The high failure rate of interspecific preganacy is a major obstacle to the successful interspectific cloning of mammals,To in vestigate the reasons for the failure of inter-specfic pregnancy between rats and mice,we transferred rat blastocysts into mouse uteri on the third day of pseudopreg -nancy (D3),oure previous study showed that intact rat embryos could still be observed in mouse uteri on D9.In the present study ,we found that expression of CD57 and CD68 increased significantly at the maternal -fetal interface fol-lowing the transfer of rat embryos,Similarly ,Leukaemia inhibitory factor(LIF) expression increased ,but vascular endothelial growth factor(VEGF) expession decreased,In a co-culture system ,the percentage of rat ectoplacental cones (EPCs) with adhesion and outgrowth and outgrowth area on mouse uterine decidual cells were less than that of mouse EPCs,These results indicate that an increase in the immunological rejection response and a decrease in the in vasiveness of rat embryos may be important reasons for the failure of interspecific pregnancy between rat and mouse.     

Interfamily pregnancy and expression of CD57, CD68 in deciduas between golden hamster and mouse

Pregnancy between different species is one of the key steps to interspecific somatic cell cloning. Although interspecific clone embryos have been constructed, they could not develop to birth after being transferred to recipi-ents. In order to clarify the mechanism of this phenomenon, interfamily pregnancy between golden hamste (Mesocricetus auratus) and mouse (Mus musculus) was studied. Co-culture results indicated that the adhesion ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 12, 24, 48 and 72 h after co-culture were all significantly lower than those of mouse blastocysts. The outgrowth ratios of golden hamster blastocysts on mouse uterine epithelia monolayer 48, 72 h after co-culture were both significantly lower than those of mouse blastocysts (P < 0.01). Golden hamster抯 blastula could be implanted and develop to D 11 of pregnancy after being transferred to mouse uterus (the 7th day after embryo transfer). Compared to the transfer of mouse embryo to mouse uterus, the successful ratio of interfamily embryo transfer was lower and the bulk of fetus was smaller than that of intraspecific fetus. Compared to intraspecific preg-nancy of mouse, the remote decidual tissue of interfamily pregnancy on D8 is looser. At the same time, expressions of CD57 and CD 68 in remote deciduas were both higher than those in the secondary deciduas in both intraspecific and interfamily pregnancy. However, expressions of the two molecules in interfamily pregnancy were lower than those in intraspecific pregnancy. These results showed that interfam-ily pregnancy could be established between golden hamster and mouse. But the development of fetus in interfamily pregnancy was slower than that in intraspecific pregnancy. The expression difference of CD57 and CD68 indicates the difference of immunoreaction between interfamily and in-traspecific pregnancy, which may be one of the reasons lead-ing to interfamily pregnancy termination.     

Global monsoon in a geological perspective

Monsoon is now considered as a global system rather than regional phenomena only. For over 300 years, monsoon has been viewed as a gigantic land-sea breeze, but now satellite and conventional observations support an alternative hypothesis which considers monsoon as a manifestation of sea-sonal migration of the intertropical convergence zone (ITCZ) and, hence, a climate system of the global scale. As a low-latitude climate system, monsoon exists over all continents but Antarctica, and through all the geological history at least since the Phenorozoic. The time is ripe for systematical studies of monsoon variations in space and time. As evidenced by the geological records, the global monsoon is controlled by the Wilson cycle on the tectonic time scale (10⁶―10⁸ a). A “Mega-continent” produces “Mega-monsoon”, and its breakdown leads to weakening of the monsoon intensity. On the time scales of 10⁴―10⁵ a, the global monsoon displays the precessional cycles of ~20 ka and eccentricity cycles of 100- and 400-ka, i.e. the orbital cycles. On the time scales of 10³ a and below, the global monsoon intensity is modulated by solar cy-cles and other factors. The cyclicity of global monsoon represents one of the fundamental factors re-sponsible for variations in the Earth surface system as well as for the environmental changes of the human society. The 400-ka long eccentricity cycles of the global monsoon is likened to “heartbeat” of the Earth system, and the precession cycle of the global monsoon was responsible for the collapse of several Asian and African ancient cultures at ~4000 years ago, whereas the Solar cycles led to the de-mise of the Maya civilization about a thousand years ago. Therefore, paleoclimatology should be fo-cused not only on the high-latitude processes centered at ice cap variations, but also on the low-latitude processes such as monsoons, as the latter are much more common in the geological his-tory compared to the glaciations.     

Optical mapping of a rice BAC clone using restriction endonuclease and imaging with fluorescent microscopy at single molecule level

A method of constructing restriction map by optical mapping and single molecule fluorescent microscopy is described. DNA molecules were aligned and adsorbed on a glass coverslip surface by a modified “molecular combing” technique, and then the surface-immobilized DNAs were cleaved in situ with a restriction endonuclease. Individual DNA molecules digested by the endonuclease EcoRⅠ were observable with fluorescent microscopy. Using optical mapping, a physical map of a rice bacterial artificial chromosome clone was constructed. This method will facilitate genomic mapping and tracing the dynamic process in real time at a single molecule level with fluorescence microscopy.     

An acidic polysaccharide with xylose branches fromPorphyra yezoensis

An acidic polysaccharide (PY3) was isolated from the hot water extract of the red algae Porphyra yezoensis by successive column chromatographies over DEAE-cellulose and Sephadex G-200. PY3 with an average molecular weight of 1.8×10⁵ was demonstrated to be composed of galactose (Gal), 3,6-anhydrogalactose (3,6-AnGal), 6-OSO₃-galactose (6-OSO₃-Gal) and xylose (Xyl) in an approximate molar ratio of 25︰15︰10︰1. In view of Smith degradation and methylation and on the basis of spectral evidence including those of IR, GC, GC-MS, and ¹H and ¹³C NMR, the most probable repeating unit of PY3 could be proposed as [(1→3)β-D-Gal(1→4)α-L-3,6-AnGal]₃-[(1→3)β-D-Gal(1→4)α-L- 6-OSO₃-Gal]₂ with a xylose moiety at the C₆ of one of every twenty-five β-D-Gal residues. To our knowledge, PY3 was shown to be the first porphyran possessing occasional xylose branches.     

Accuracy of a combined score of zygote and embryo morphology for selecting the best embryos for IVF

Objective： To evaluate the accuracy of a scoring system combining zygote and embryo morphology in predicting the outcome of in vitro fertilization （IVF） treatment. Methods： In a study group, 117 consecutive IVF or intracytoplasmic sperm injection （ICSI） cycles with embryo transfer were carried out and 312 embryos were scored using a combined scoring system （CSS） of zygote and embryo morphology before transplantation. In a control group, a total of 420 IVF or ICSI cycles were carried out and 1176 embryos were scored using a cumulative embryo score （CES）. The effects of the combined scoring system on the embryo implantation rate and pregnancy rate per cycle were analyzed. Results： Using the combined scoring system, the embryo implantation rate （27.6%） and the clinical pregnancy rate （48.7%） were significantly higher than those in the control group （20.8% and 38.6%, respectively）. Also, the implantation rate of embryos scoring ≥70 （38.5%： 82 sacs/213 embryos） was significantly higher （P〈0.001） than that of embryos scoring 〈70 （4%： 4 sacs/99 embryos）. The pregnancy rate of patients with embryos scoring ≥70 using the combined scoring system （66.7%） was significantly higher （P〈0.001） than that of patients with embryos scoring ≥20 using the cumulative embryo score （59.0%）. Conclusion： The results suggest that selecting embryos with a high score （≥70） using the combined scoring system could increase the implantation rate and pregnancy rate, and that using a scoring system combining assessments of human zygotes and pre-implantation embryos might predict IVF outcomes more accurately than using a cumulative embryo score.     

Morphological properties of nociceptive and non-nociceptive neurons in primary somatic cerebral cortex (SI) of cat

With the techniques of intracellular recording and labelling, we investigated pain sensation and modulation of the somatic cortical cortex at the neuron’s level. After observing the evoked potentials from stimulating the saphenous nerves (SN) of 654 neurons in SI area of the cats, we labelled 30 of the neurons with Neurobiotin to preserve the distribution and the morphologic characteristics of the neurons in the cortex. Based on the tridimensional reconstruction in addition to the eletrophysiological functions, we found clear morphological distinctions between nociceptive and non-nociceptive neurons (P＜0.01). This result provided new experimental material to illustrate the function of nociceptive neurons in somatosensory cortex (SI) and presented further evidence to support the “specificity theory” of pain sensation in terms of morphology.     

Effect of UPP on the expression of VEGF and its receptors in mouse uterus during peri-implantation

On day 3 of gestation ,one uterine horn of female pregnant mouse was injected intraluminally with 5 μL 0.1μg/mL lactacystin,a specific inhibitor of ubiquitin-pro-teasome pathway (UPP),while the contralateral horn served as control ,Animals were sacrificed by cervical dislocation on day 5,6,7 of gestation ,respectively,Then the number of implanted embryos in each uterine horn was calcuated,and the expression of VEGF and its receptors was examined,The data showed that the number of implanted embryos was decreased significantly after treatment with lactacystin ,The results of RT-PCR and Western blot indicated that expression of VEGF and its receptors at mRNA and protein levels was significantly decreased in the treated uterus,menawhile,the expression of HIF-1α(the α subunit of HIF ,a transcrip-tional factor of VEGF) was reduced at both mRNA and protein levels,These data suggested that the effect of UPP on VEGF expression was realized through regulating HIF-1α expression .In addition ,UPP is likely to take part in the modulation of VEGF receptors expression ,These changes may be one of the reasons for the reduction of implanted embryos.     

Comparative study on the effect of integrin αvβ3 on secretion of matrix metalloproteinases in human normal and carcinoma trophoblasts

The invasion of cytotrophoblast cells into the maternal endometrium during embryonic implantation is very similar to the metastasis of carcinoma cells. However, the significant difference is that the former is a highly controlled process. In this report, the effects of integrin αvβ3 on the secretion of matrix metalloproteinase (MMP) were compared between normal cytotrophoblast cells and choriocarcinoma cells by RT-PCR, gelatin zymography and immunocytochemistry. The results reveal that both normal cytotrophoblast cells and choriocarcinoma cells can express integrin αvβ3. The secretion of MMPs in normal cytotrophoblast cells is up-regulated by anti-αvβ3 antibody, whereas, decreased in choriocarcinoma cells. It was suggested that αvβ3 can modulate the expression of MMPs in trophoblasts, and this action is carried out through distinct mechanisms in normal and carcinoma cytotrophoblast cells.     

Aberrant DNA methylation patterns in cultured mouse embryos

Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.     

Evidence for cold events in the early Holocene from the Guliya ice core, Tibetan Plateau, China

Evidence for the “8.2 ka cold event” has been provided mostly from the circum-North Atlantic area. However, whether this cold event occurred in other places is a key to understanding its cause. Here, we provide the evidence for the “8.2 ka cold event” from the Guliya ice core in the northwest Tibetan Plateau, and it was found that the peak cooling (~8.3—8.2 ka) in this ice core was about 7.8—10℃, which was larger than the cooling in the North Atlantic region. The primary causes for this episode were diminished solar activity and weakened thermohaline circulation. Moreover, another weak cold event, centered about 9.4 ka, was also recorded in the Guliya ice core record. These two cold events were concurrent with the ice-rafting episodes in the North Atlantic during the early Holocene, which implies that the millennial-scale climatic cyclicity might exist in the Tibetan Plateau as well as in the North Atlantic.     

热轧和退火温度对SP-700合金组织和力学性能的影响

The effect of rolling temperature on both two- and single-phase regions and annealing in a temperature range of 700–950°C on the microstructure and mechanical properties of Ti?5Al?4V?2Fe?1Mo alloy was investigated. The results indicated that the best balance of strength and ductility is obtained by rolling in the two-phase region due to the globularization of the alpha phase and increase in its volume fraction. After rolling in the two-phase region, the ductility of the specimens annealed at 700 to 800°C increased because of the finer size and globularized alpha phase, while the reduction in strength was attributed to a decrease in the alpha phase volume fraction. However, at 950°C, the strength increased and ductility dropped by the formation of acicular alpha phase due to an increase in the phase boundary area. Annealing and aging after rolling in the beta-phase region increased the strength and decreased the ductility, which is attributed to the formation of a secondary alpha phase. A combination of favorable yield strength (1113 MPa) and elongation (13.3%) was obtained through rolling at 850°C followed by annealing at 750°C and aging at 570°C.     

Positional variation of antipodal cells in polyembryonic rice ApⅢ before and after fertilization

The variation in the position of antipodal cells before and after fertilization in polyembryonic rice strain ApⅢ was studied by using conventional sectioning technique. It was shown that initially the three antipodal cells lie at the chalazal end of the young embryo sacs. Along with the development of embryo sac, the antipodal cells proliferate into a multicellular structure containing up to about 20 cells. Most of the cells migrate to its dorsal side when the embryo sacs turn into mature. In a few embryo sacs the antipodal cells are situated in the positions adjacent to the egg apparatus at the micropylar end, while in other sacs, they spread from the midst of the embryo sac to the micropylar end clinging to the nucellar tissue. Furthermore, it is reported for the first time that cells of some egg apparatus or proembryos disorganize when antipodal cells lie at the micropylar end, indicating that the function of the antipodal cells may vary during the embryogenesis in rice ApⅢ.     

Sources of cumulus expansion enabling factor (CEEF) in porcine follicles

It was shown that expansion of porcine cumulus did not depend on oocyte-secreted factor(s), and it is therefore presumed that porcine CEEF may not be produced exclusively by the oocyte. In this experiment, we used mouse oocytectomized complexes (OOX), which were incapable of CEEF production, to assess the secretion of CEEF by evacuated zona, oocytes of different quality and somatic cells in the porcine follicles. The results showed that: (ⅰ) Evacuated zonae from both porcine and mouse oocytes did not produce CEEF. (ⅱ) Porcine oocytes of A, B and C types from 3—6 mm follicles were not significantly different in both production and activity of CEEF. (ⅲ) Both porcine OOX from 3—6 mm follicles and granulose cells from < 1 mm follicles secreted CEEF in a large quantity, independent of gonadotropins; mural granulose cells from 3—6 mm follicles, however, produced neglectable amount of CEEF. (ⅳ) The follicular fluid from 3—6 mm porcine follicles contained CEEF activity that was concentration-dependent, and thus it enabled cumulus expansion in 60% mouse OOX when used at 10% of concentration, but the expansion rate of mouse OOX decreased to 9% when the concentration was increased to 50%. (ⅴ) Mouse OOX cultured in porcine CEEF-containing M199 expanded only in the presence of gonadotropins, suggesting that the activity of porcine CEEF is hormone-dependent.