Maternal age effect on early human embryonic development and blastocyst formation

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([expanded blastocysts/blastocysts]*100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (<30 years, 82%; ≥40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. © 1996 Wiley-Liss, Inc.     

Assembly and tissue functions of early embryonic laminins and netrins

Vertebrate laminins and netrins share N-terminal domain structure, but appear to be only distantly related. Both families can be divided into different subfamilies on the basis of structural considerations. Recent observations suggest that specific laminin and netrin members have developmental functions that are highly conserved across species. Vertebrate laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1), like the two Caenorhabditis elegans laminins, are embryonically expressed and are essential for basement membrane assembly. Basement membrane assembly is a cooperative process in which laminins polymerize through their LN domains and anchor to the cell surface through their G domains; this leads to cell signaling through integrins and dystroglycan (and possibly other receptors) recruited to the adherent laminin. Netrins may associate with this network through heterotypic LN domain interactions. Vertebrate netrin-1, like invertebrate UNC-6/netrins, is well known as an extracellular guidance cue that directs axon migration towards or away from the ventral midline. It also regulates cell adhesions and migrations, probably as a basement membrane component. Although sharing structural features, these two vertebrate protein families are quite distinct, having both retained members that mediate the ancestral developmental functions.     

From blastocyst to gastrula: gene regulatory networks of embryonic stem cells and early mouse embryogenesis

To date, many regulatory genes and signalling events coordinating mammalian development from blastocyst to gastrulation stages have been identified by mutational analyses and reverse-genetic approaches, typically on a gene-by-gene basis. More recent studies have applied bioinformatic approaches to generate regulatory network models of gene interactions on a genome-wide scale. Such models have provided insights into the gene networks regulating pluripotency in embryonic and epiblast stem cells, as well as cell-lineage determination in vivo. Here, we review how regulatory networks constructed for different stem cell types relate to corresponding networks in vivo and provide insights into understanding the molecular regulation of the blastocyst–gastrula transition.     

Engineered early embryonic cardiac tissue retains proliferative and contractile properties of developing embryonic myocardium

Embryonic myocardium has a high rate of cell proliferation and regulates cellular proliferation, contractile function, and myocardial architecture in response to changes in external mechanical loads. However, the small and complex three-dimensional (3D) structure of the embryonic myocardium limits our ability to directly investigate detailed relationships between mechanical load, contractile function, and cardiomyocyte proliferation. We developed a novel 3D engineered early embryonic cardiac tissue (EEECT) from early embryonic ventricular cells to test the hypothesis that EEECT retains the proliferative and contractile properties of embryonic myocardium. We combined freshly isolated White Leghorn chicken embryonic ventricular cells at Hamburger-Hamilton (HH) stage 31 (day 7 of a 46-stage, 21-day incubation period), collagen type I, and matrix factors to construct cylindrical-shaped EEECTs. We studied tissue architecture, cell proliferation patterns, and contractile function. We then generated engineered fetal cardiac tissue (EFCT) from HH stage 40 (day 14) fetal ventricular cells for direct comparison with EEECT. Tissue architecture was similar in EEECT and EFCT. EEECT maintained high cell proliferation patterns by culture day 12, whereas EFCT decreased cell proliferation rate by culture day 9 (P < 0.05). EEECT increased active contractile force from culture day 7 to day 12. The culture day 12 EEECT contractile response to the beta-adrenergic stimulation was less than culture day 9 EFCT (P < 0.05). Cyclic mechanical stretch stimulation induced myocardial hyperplasia in EEECT. Results indicate that EEECT retains the proliferative and contractile properties of developing embryonic myocardium and shows potential as a robust in vitro model of developing embryonic myocardium.     

Rates of oxygen uptake by embryonic anterior horn tissue isolated at various developmental stages.

Neuroepithelial cells of the presumptive spinal cord (stage 11) consume oxygen, albeit at a low rate. As neurons differentiate in the presumptive motor horns the rate of oxygen consumption increases to approximately 70 mumoles/g wet wt/hr by stage 26. It is suggested that the rate of oxygen consumption per unit volume of neuron then remains constant as subsequent development ensues but since the neurons become more widely spaced the oxygen consumption per unit volume of anterior horn tissue decreases.     

Role of embryonic oestrogen in rabbit blastocyst development and metabolism

Rabbit morulae were grown for 24 h in Ham's F12 medium supplemented with BSA. CI-628 citrate (1.5 micrograms/ml), a specific oestrogen antagonist, significantly inhibited the transformation of morulae to blastocysts. This inhibition was reversed with oestradiol-17 beta (1 micrograms/ml) but not oestradiol-17 alpha (1 micrograms/ml) added to the culture medium. The specific activities of phosphofructokinase, lactic dehydrogenase, malate dehydrogenase and alkaline phosphatase in blastocysts grown in vitro for 24 h in medium TC 199 + BSA showed significant elevation with blastocyst growth and expansion, while that of acid phosphatase revealed no change, and leucine aminopeptidase activity declined significantly. These changes were markedly inhibited by CI-628 citrate (2 micrograms/ml) and were reversed by oestradiol-17 beta (0.4 micrograms/ml) but not by oestradiol-17 alpha (0.4 micrograms/ml). Our findings suggest a role of oestrogen present in the rabbit morula and blastocyst in the triggering of embryonic differentiation and metabolic functions.     

A caprine chimera produced by injection of embryonic germ cells into a blastocyst

This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28-42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo.     

Induction of virulence response in Agrobacterium tumefaciens by tissue explants of various plant species

Forty-four plant species belonging to different taxa were tested for their ability to induce the expression of the virulence E gene in Agrobacterium tumefaciens containing virE:lacZ fusion constructs. With the exception of 6 algae, one fern and 2 monocots, tissue explants of all other plants (2 Algae, 3 Bryophytes, 2 Pteridophytes, 15 Gymnosperms, 8 Monocots and 5 Dicots) induced the expression of the virE gene as detected by the presence of -galactosidase activity in the bacteria.Abbreviations AS acetosyringone - vir virulence genes Scientific Contribution Number 1734 from the New Hampshire Agricultural Experiment Station     

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

Oestrogen receptors and survival in early breast cancer.

Oestrogen receptor status was related to survival in 414 patients with primary breast cancer. Women with oestrogen receptors in their tumours survived significantly longer than those without receptors; this was true for both premenopausal and postmenopausal women and also when the patients were subdivided into those with and without axillary metastases. Patients with axillary metastases and no oestrogen receptors in their tumours had the worst prognosis, while women with axillary metastases and oestrogen receptors had a death rate similar to that of women with no axillary metastases and no receptors. Patients without oestrogen receptors and with no axillary metastases were identified as a high-risk group, and it would seem appropriate to include such patients in future trials for adjuvant therapy in early breast cancer.     

Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)