Identification of perivascular and stromal mesenchymal stem/progenitor cells in porcine endometrium.

Mammalian uterus contains a population of mesenchymal stem/progenitor cells that likely contribute to endometrial regeneration during each reproductive cycle. In human and mouse, they reside in perivascular, epithelial and stromal compartments of the endometrial functionalis and basalis. Here, we aimed to identify tissue resident cells expressing mesenchymal stem cell markers CD29, CD44, CD90, CD105, CD140b and CD146 in the porcine endometrium. We used single immunofluorescence and Western blotting. Each of these markers was detected in small cells surrounding endometrial blood vessels. CD105 and CD146 were also expressed in single stromal cells. A few stromal and perivascular cells showed the presence of pluripotency marker Oct4 in the cytoplasm, but not in the nucleus, which may imply they are not truly pluripotent. Endometrial cell cultures were examined for the expression of CD29, CD44, CD90, CD105 and CD140b proteins and tested in wound-healing assay and culture model of chemotaxis. In conclusion, our results demonstrate perivascular location of prospective mesenchymal stem/progenitor cells in the porcine endometrium and may suggest that stromal CD105+ and CD146+ cells represent more mature precursors originating from their perivascular ancestors.

First Report of Alfalfa mosaic virus Infecting Tedera (Bituminaria bituminosa (L.) C.H. Stirton var. albomarginata and crassiuscula) in Australia.

Tedera (Bituminaria bituminosa (L.) C.H. Stirton vars albomarginata and crassiuscula) is being established as a perennial pasture legume in southwest Australia because of its drought tolerance and ability to persist well during the dry summer and autumn period. Calico (bright yellow mosaic) leaf symptoms occurred on occasional tedera plants growing in genetic evaluation plots containing spaced plants at Newdegate in 2007 and Buntine in 2010. Alfalfa mosaic virus (AlMV) infection was suspected as it often causes calico in infected plants (1,2) and infects perennial pasture legumes in local pastures (1,3). Because AlMV frequently infects Medicago sativa (alfalfa) in Australia and its seed stocks are commonly infected (1,3), M. sativa buffer rows were likely sources for spread by aphids to healthy tedera plants. When leaf samples from plants with typical calico symptoms from Newdegate (2007) and Buntine (2010) were tested by ELISA using poyclonal antisera to AlMV, Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV), only AlMV was detected. When leaf samples from 864 asymptomatic spaced plants belonging to 34 tedera accessions growing at Newdegate and Mount Barker in 2010 were tested by ELISA, no AlMV, BYMV, or CMV were detected, despite presence of M. sativa buffer rows. A culture of AlMV isolate EW was maintained by serial planting of infected seed of M. polymorpha L. (burr medic) and selecting seed-infected seedlings (1,3). Ten plants each of 61 accessions from the local tedera breeding program were grown at 20°C in an insect-proof air conditioned glasshouse. They were inoculated by rubbing leaves with infective sap containing AlMV-EW or healthy sap (five plants each) using Celite abrasive. Inoculations were always done two to three times to the same plants. When both inoculated and tip leaf samples from each plant were tested by ELISA, AlMV was detected in 52 of 305 AlMV-inoculated plants belonging to 36 of 61 accessions. Inoculated leaves developed local necrotic or chlorotic spots or blotches, or symptomless infection. Systemic invasion was detected in 20 plants from 12 accessions. Koch's postulates were fulfilled in 12 plants from nine accessions (1 to 2 of 5 plants each), obvious calico symptoms developing in uninoculated leaves, and AlMV being detected in symptomatic samples by ELISA, inoculation of sap to diagnostic indicator hosts (2) and RT-PCR with AlMV CP gene primers. Direct RT-PCR products were sequenced and lodged in GenBank. When complete nucleotide CP sequences (666 nt) of two isolates from symptomatic tedera samples and two from alfalfa (Aq-JX112758, Hu-JX112759) were compared with that of AlMV-EW, those from tedera and EW were identical (JX112757) but had 99.1 to 99.2% identities to the alfalfa isolates. JX112757 had 99.4% identity with Italian tomato isolate Y09110. Systemically infected tedera foliage sometimes also developed vein clearing, mosaic, necrotic spotting, leaf deformation, leaf downcurling, or chlorosis. Later-formed leaves sometimes recovered, but plant growth was often stunted. No infection was detected in the 305 plants inoculated with healthy sap. To our knowledge, this is the first report of AlMV infecting tedera in Australia or elsewhere. References: (1) B. A. Coutts and R. A. C. Jones. Ann. Appl. Biol. 140:37, 2002. (2) E. M. J. Jaspars and L. Bos. Association of Applied Biologists, Descriptions of Plant Viruses No. 229, 1980. (3) R. A. C. Jones. Aust. J. Agric. Res. 55:757, 2004.

Lipid content and cryotolerance of porcine embryos cultured with phenazine ethosulfate.

The addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on pig embryo development, apoptosis, cytoplasmic lipid content and survival after OPS vitrification. Porcine zygotes were cultured in NCSU-23 medium supplemented with 0 (control) or 0.05 microM PES up to the blastocyst stage and were vitrified using OPS technology. Culture of embryos with PES reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (45.2 and 37.9 percent, respectively). These results showed that culturing porcine embryos in medium with PES increased the proportion of morula and blastocyst formation and reduced the index of DNA fragmentation and the cytoplasmic lipid content of cultured blastocysts. However, the use of PES during in vitro culture had limited effect on porcine blastocyst survival after vitrification.

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos.

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.

Changes of lipid composition in non-cultured and cultured porcine embryos.

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.

Species differences in respiratory rhythm generation in rodents.

We examined the role of riluzole (RIL)- and flufenamic acid (FFA)-sensitive mechanisms in respiratory rhythmogenesis in rats and hamsters using the in situ arterially perfused preparation. Based on the hypothesis that respiratory networks in animals capable of autoresuscitation would have a greater prevalence of membrane mechanisms that promote endogenous bursting, we predicted that older (weaned) hamsters (a hibernating species) would be more sensitive to the blockade of RIL- and FFA-sensitive mechanisms than age-matched rats and that younger (preweaned) rats would behave more like hamsters. Consistent with this, we found that respiratory motor output in weaned hamsters [>21 days postnatal (P21)] was highly sensitive to RIL (0.2-20 muM), while in young rats (P12-14) it was less so (only affected at higher concentrations of RIL), and weaned rats were not affected at all. On the other hand, respiratory motor output was equally reduced by FFA (0.25-25 muM) in both young and weaned rats but was unaffected in weaned hamsters. Coapplication of RIL and FFA (RIL + FFA) produced greater inhibition of respiration in both young and weaned rats compared with either drug alone. In contrast, in weaned hamsters, FFA coapplication offset the inhibitory effect of RIL alone. Increasing respiratory drive with hypercapnia/acidosis ameliorated the respiratory inhibition produced by RIL + FFA in weaned rats but had no effect in young rats. Data from the present study indicate that respiratory rhythmogenesis in young rats is more dependent on excitatory RIL-sensitive and FFA-sensitive mechanisms than older rats and that fundamental differences exist in the respiratory rhythmogenic mechanisms between rats and hamsters.

Lipid content of non-cultured and cultured pig embryo.

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos. The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage. Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume - V(e), volume density of cytoplasm per unit volume of embryo - Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm - Vv(fat,c) and total volume of lipid droplets per whole embryo - V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

[Potter sequence in a neonate with bilateral renal dysplasia with genetic aspects]. / Sekwencja potter u noworodka z obustronna dysplazja nerek w aspekcie genetycznym.

The authors describe a neonate with Potter sequence due to bilateral renal dysplasia and discuss its genetic implications. Although this congenital anomaly of the kidneys may occur sporadically, it is now accepted that this condition has an autosomal dominant inheritance pattern with a penetrance of 50-90% and variable expression. Potter sequence is not compatible with extrauterine life; therefore, in case of maternal oligohydramnios, ultrasound should be performed in the second trimester in order to make a prenatal diagnosis, as well as to permit genetic counseling. As Potter sequence is frequently associated with clinically silent anomalies of the kidneys, ultrasound examination of the kidneys and urinary tract should also be performed in all family members.

[Criminal contamination by metallic mercury of cow's silage]. / Kryminalne skazenie rtecia metaliczna paszy dla krów.

This paper presents an unsuccessful homicidal attempt on 23 persons by means of metallic mercury added in large amount to cow's silage. Thirteen adults and twelve children used milk from these cows for 4.5 months. The mercury concentration in the milk of these cows as well as in serum and daily urine of consumers of the milk were examined by atomic absorption spectrophotometry. Despite the increased level of mercury in the milk (average 0.023 mg/kg), the concentrations of mercury in daily urine of only three persons were raised about 50%. These people had been working in a cowshed without ventilation every day for many months and thus had been exposed to vaporised mercury.

Survival of pig morula and blastocyst after exposure to vitrification media or vitrification.

The aim of this experiment was to examine the survival of porcine embryos following exposure to vitrification solutions and vitrification. The work was carried out on non-cultured and cultured morulae and blastocysts. The viability of treated embryos was assessed by their ability to develop in in vitro culture. The results showed that the most detrimental step in the vitrification of pig embryos is exposing them specifically to a vitrification medium rather than the vitrification process itself. Moreover, we demonstrated the beneficial effect of culture on the viability of pig embryos after vitrification

Cryopreservation of mammalian ova and embryos by vitrification.

Vitrification is a new approach to oocyte and embryo cryoconservation. It consists in the solidification of a solution caused not by crystallization, but by a drastic increase in viscosity during cooling. The application of this approach to cryoconservation of oocytes and embryos of different species depends upon the development of proper procedures and non-toxic media. From the technical point of view, the vitrification method is simple and relatively easily applicable under field conditions. The authors review the current procedures applied to oocytes and embryos of laboratory and farm animals.

Factors affecting the survival of one- and two-cell rabbit embryos cryopreserved by vitrification.

The effects of equilibration time, glycerol (GLY), and 1,2-propanediol (PROH) concentration, and of vitrification and sucrose solution on the viability of 1- and 2-cell rabbit embryos were investigated. After collection, the embryos were equilibrated for 5 or 10 minutes in phosphate buffered saline (PBS) containing 10% GLY-20% PROH and were exposed for 30 seconds at 4 degrees C or were exposed and vitrified in one of two vitrification solutions 35% GLY-35% PROH or 20% GLY-50% PROH. The in vitro survival rates of 1-cell embryos equilibrated for both 5 and 10 minutes were lower (34.0 and 48.0%, respectively) than those of 2-cell embryos (78.8 and 68.5%, respectively; P<0.01). No differences were noted in the viability of embryos exposed to the 2 vitrification solutions. Following vitrification in a mixture of 35% GLY-35% PROH, the survival rates of 1- and 2-cell embryos were 18.3 and 13.7% and 19.6 and 10.4% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1- and 2-cell embryos vitrified in a solution of 20% GLY-50% PROH were 25.7 and 35.4% and 26.2 and 21.3% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1-and 2-cell embryos stored in 1M sucrose solution were 63.8 and 84.0%, respectively. In conclusion, the viability of vitrified 1- and 2-cell rabbit embryos was reduced as a consequence of their equilibration before vitrification, the exposure to vitrification solution and the dilution in a sucrose solution rather than of the vitrification process itself.

The effect of sucrose and trehalose on viability of one- and two-cell rabbit embryos.

The effect of sucrose and trehalose on the viability of one- and two-cell rabbit embryos was investigated. A significant decrease in the viability of one- and two-cell embryos exposed for 30 min. at 20 degrees C was observed. At 38 degrees C none of the two-cell embryos in a sucrose solution survived after 30 min exposure, while approximately 50% of the embryos survived in a trehalose solution. The cleveage rate in culture of two-cell embryos exposed both to 2.0 M or 1.45 M trehalose was significantly lower in comparison with the control group. However the survival rate after transfer of two-cell embryos exposed to 1.45 M trehalose solution at 20 degrees C remained the same as that of the control group.

Stage-dependent viability of vitrified rabbit embryos.

The aim of the work was to determine the susceptibility of rabbit embryos to vitrification at different developmental stages. The experiment was carried out on 676 embryos at 1-, 2- and 8-to 16-cell stages as well as the morula and blastocyst stages. As a vitrification medium, a mixture of 30% 1,2-propanediol + 30% glycerol (Solution I), or 35% 1,2-propanediol + 35% glycerol (Solution II), was used. The embryos were frozen in glass ampules placed in nitrogen vapour for 5 min before being plunged into liquid nitrogen. Dilution after rapid thawing was done in one step in a 1-M sucrose solution. After vitrification in Solution I, none of the 1- or 2-cell embryos survived, whereas the survival rate of 8-to 16-cell embryos, morula and blastocysts, was 23.0, 82.7 and 78.5%, respectively. After vitrification in Solution II, the survival rate of 1-, 2- and 8-to 16-cell embryos was 20.0, 43.8 and 92.9%, respectively. The proportion of live offspring on the Day 28 after transfer of 68 vitrified morula was 26.5% compared with 24.0% in the control group. Thus, the proposed vitrification procedures can be useful in the cryopreservation of rabbit embryos.

Effect of dexamethasone and hydrocortisone on the course of superovulation in cattle.

The effect of hydrocortisone and dexamethasone on superovulation was examined in 12 cows. On the day PMSG was given, each animal received either the first of five daily doses of 250 mg succinate hydrocortisone or one injection of 30 mg dexamethasone. In the 48-hr interval between the injection of PMSG and PGF(2)alpha, the concentration of progesterone rose from 6.97 to 10.22 ng/ml in the experimental groups and only to about 2.8 ng/ml in the control group. In the following days progesterone increased even more, from 15.7 to 26.0 ng/ml seven days after estrus in the experimental group and to 19.25 ng/ml in the control group. The group which received dexamethasone had an average of 4.7 corpora lutea and one embryo flushed per animal. The hydrocortisone group had an average of 2.5 corpora lutea and one cow had two embryos. The control group had 6.2 corpora lutea and 5.2 embryos per animal.