Summary. Dispersed cells from cultured mouse cell lines, mouse macrophages, and inert microspheres were layered onto outgrowing mouse trophoblast in culture. The cells that settled onto the trophoblast remained round, in contrast to the elongated spreading shape they assumed on the glass substratum. The cells were readily dislodged from the trophoblast surface, whereas the microspheres were strongly adherent to trophoblast within 30 min. Scanning electron microscopy showed that trophoblast engulfed the spheres, but not the cells. Despite the lack of adhesion between cells and trophoblast, cell processes connected the two. The inability of cells to adhere to the free surface of the trophoblast could explain the trophoblast's ability to induce contact inhibition in co-cultured cells.
R. H. Glass, A. I. Spindle, M. Maglio and R. A. Pedersen
Y Du, C S Pribenszky, M Molnár, X Zhang, H Yang, M Kuwayama, A M Pedersen, K Villemoes, L Bolund and G Vajta
The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4±2.4%) or 130 (13.1±3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 °C or 25 °C. Oocytes pressurized at 37 °C had a significantly higher blastocyst (14.1±1.4%) rate than those treated at 25 °C (5.3±1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.
A Stronati, G C Manicardi, M Cecati, M Bordicchia, L Ferrante, M Spanò, G Toft, J P Bonde, B A G Jönsson, A Rignell-Hydbom, L Rylander, A Giwercman, H S Pedersen, E C Bonefeld-Jørgensen, J K Ludwicki, V Lesovoy, D Sakkas and D Bizzaro
Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652 adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p′-DDE), as a proxy of the total DDT exposure were determined. Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting pro-apoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas our study failed to demonstrate any relations between DDE and %TUNEL positivity and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall regions. These results suggest that CB-153 and related chemicals might alter sperm DNA integrity and Bcl-xL levels in European adult males, but not in the highly exposed Inuit men. Additional issues (genetic background, lifestyle habits and characterization of total xeno-hormonal activities) need to be investigated in order to fully assess the population variations observed.
R Michael Roberts, Kyle M Loh, Mitsuyoshi Amita, Andreia S Bernardo, Katsuyuki Adachi, Andrei P Alexenko, Danny J Schust, Laura C Schulz, Bhanu Prakash V L Telugu, Toshihiko Ezashi and Roger A Pedersen
It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.