New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n?=?16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n?=?16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

     

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.     

Adaptive ultrastructural changes in soil-resident Yersinia pseudotuberculosis bacteria

After the prolonged residence (from 1 month to 2 years) in flow soil columns at 6 - 8 degrees C and 18 - 20 degrees C a complex of ultrastructural changes was detected in Y. pseudotuberculosis bacteria, depending on temperature, the duration of residence in the soil and, to a definite extent, on the strain. They were manifested in the form of cell-wall changes, the formation of the capsule and intercellular slime, changes in the ribosomal saturation of cytoplasm and the conformation state of DNA in the nucleoid zone. As the result of adaptation to nutritional deficit storage substances were accumulated in the form of electron-dense inclusions (polyphosphates) and pseudovacuoles (poly-beta-oxibutyric acid). Temperature influenced the process of mitotic division and the state of chromatin in bacteria. The described ultrastructural changes Y. pseudotuberculosis may be regarded as natural adaptive reaction to the altered conditions of their residence.     

Influenza activity in Czechoslovakia and the USSR, 1980-1985

Between 1980 and 1985, Czechoslovakia had experienced 4 and the USSR 3 major influenza outbreaks. Of the 3 epidemic outbreaks in the USSR, 2 were associated with influenza B virus (in the 1980/81 and 1983/84 seasons) and 1 with influenza A virus of the H3N2 subtype. In the USSR, influenza A (H1N1) virus never predominated as a cause of epidemic during the 5 years period. In Czechoslovakia, 2 epidemics (in the 1980/81 and 1983/84 seasons) were due to influenza A (H1N1) virus. The epidemic in the 1981/82 season had two waves of unequal heights and a mixed type B and subtype A (H3N2) etiology; a two-wave epidemic associated with isolates of influenza A (H1N1) and influenza B viruses was also recorded in the 1983/84 season. The influenza A (H3N2) epidemic in 1983 was of explosive character. All influenza viruses circulating in the two countries between 1980 and 1985 were of the same antigenic profile, but were isolated from the epidemics that occurred in different influenza seasons. The virological surveillance revealed strains of virus closely related to drift variants detected from outbreaks in 1977-1979 and the new variants A/Chile 1/83, A/Philippines 2/82, A/Caen 1/84 and B/USSR 100/83.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Influence of temperature on the variability of Listeria monocytogenes in the course of its prolonged existence in soil-packed water-circulating columns

The capacity of L. monocytogenes for prolonged existence in the soil at different temperatures (18-20 degrees C and 6-8 degrees C) has been shown. The viability of these bacteria in the soil depends both on their properties and the temperature factor. Low temperatures (6-8 degrees C) ensure, on the whole, the stable preservation of the biological properties of the bacteria. More pronounced variability of L. monocytogenes at a temperature of 18-20 degrees C is an adaptive property, ensuring high ecological plasticity of the bacteria and their prolonged existence in the soil.     

Congenitally caused fused labia in the common marmoset (Callithrix jacchus)

In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.     

The gonad maturation and size structure of the population of abundant planktonic predator <Emphasis Type="Italic">Eukrohnia hamata</Emphasis> (Möbius, 1875) (Chaetognatha) in the Eurasian basin of the Arctic Ocean in summer

The size structure of the population, morphology of the reproductive system, and gonad maturation of the chaetognath Eukrohnia hamata (Möbius, 1875) (Chaetognatha) were studied for the first time from zooplankton samples collected in Fram Strait and the deep Eurasian basin of the Arctic Ocean north of the Kara and Laptev seas in the summers of 1995 and 1997. We describe the gonad morphology and the progress of gonad maturation in male and female specimens and propose a system for classification of gonad maturity stages. During the Arctic summer, the population was represented by individuals ranging in size from 2 to 37 mm. The newly hatched young (2.0–3.5 mm long) and juveniles (4.0–12.5 mm long) dominated numerically (30 and 50% of the population, respectively). Our results indicate that E. hamata successfully matures and reproduces in the Arctic basin, but mature individuals make up less than 1% of the population in the summer against the background of juvenile abundance.     

Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness

The aim of the present investigations was to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryoinjuries. Spermatozoa were isolated and vitrified using five different mediums: Group 1: standard buffer for fish spermatozoa, Cortland(?)-medium (CM, control); Group 2: CM+1% bovine serum albumin (BSA); Group 3: CM+1% BSA+0.125 M sucrose; Group 4: CM+1% BSA+40% seminal plasma; and Group 5: CM+1% BSA+40% seminal plasma+0.125 M sucrose. For cooling, 20 μL suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility, cytoplasmic membrane integrity (SYBR-14/propidium iodide staining technique), and mitochondrial membrane integrity (JC-1 staining). Motility (86%, 71%, 80%, 81%, and 82%, for Groups 1, 2, 3, 4, and 5, respectively) and cytoplasmic membrane integrity (90%, 82%, 83%, 84%, and 87%, respectively) of spermatozoa in all the 5 groups were not decreased significantly. All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility and cytoplasmic membrane integrity. However, mitochondrial membrane potentials of the spermatozoa in Groups 1, 2, 3, 4, and 5 were changed significantly (6%, 50%, 37%, 55%, and 34%, respectively) (P(1,2,3,4,5)<0.001; P(2,3,4,5) <0.01)(P(3-5)>0.1). This rate was maximal in Group 4 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma without significant loss of important physiological parameters.