Elena Kizilova

Preimplantation mouse embryo development as a target of the pesticide methoxychlor

Effects of methoxychlor (MXC) and estradiol-17beta (E) were studied in mouse preimplantation embryos. Pregnant mice received s.c. injections of sesame oil only, 10 microg E, or 0.5 mg purified (95%) MXC on Days 2-4 of pregnancy (plug = Day 1). Another group received a single dose of 2.5 microg E on Day 2 only. Based on the average weight of pregnant females, 10 microg of estradiol was equivalent to 0.33 mg/kg of bw, 2.5 microg of estradiol was equivalent to 0.082 mg/kg of bw, and the 0.5-mg dose of MXC was equivalent to 16.5 mg/kg of bw. All embryos were collected for analyses on Day 4. MXC and both estradiol-17beta doses suppressed embryonic development to blastocyst, decreased embryo cell numbers, and caused abnormal blastocyst formation. The high estradiol-17beta dose significantly increased the percent degenerating embryos and caused a tube-locking effect, with retention of embryos in the oviduct. In contrast to estradiol-17beta, MXC at the dose used in this study did not alter tubal transport of embryos. Also in contrast to estradiol-17beta, MXC increased the percentage of nuclear fragmentation and micronuclei. In preimplantation embryos, MXC and estradiol-17beta both suppressed embryo development. MXC effects were, however, different from those of estradiol-17beta, indicating a difference in mechanism of action, possibly due to cytotoxicity and induction of apoptosis.

Stem cells in the reproductive strategy of colonial rhizocephalan crustaceans (Crustacea: Cirripedia: Rhizocephala)

Summary In vivo, histological, histochemical, immunochemical and ultrastructural investigations were performed of the colonial internae of Peltogasterella gracilis, Polyascus (Sacculina) polygenea, and Thylacoplethus isaevae (all infesting decapods). It was shown that asexual reproduction in these species occurs through the budding of stolon-like structures. Undifferentiated stem cells were found inside the stolons. They take part in the morphogenesis of the earliest buds, and later migrate to the developing ovary as primary germ cells. The stem cells in P. polygenea, P. gracilis and Th. isaevae selectively express alkaline phosphatase activity, a well known histochemical marker for mammalian embryonic stem cells in vivo and in vitro. It is shown in P. gracilis that stem cells also selectively express proliferating cell nuclear antigen (PCNA), a cellular marker for cell reproduction. The reproductive strategy in colonial rhizocephalans includes a three-level cascade: asexual reproduction by budding in interna; repeated development of multiple externae; and, in some cases, repeated cycles of sexual reproduction in each externa, resulting in large numbers of hatching larvae and allowing the parasite to infest a large proportion of the host population. Thus, sexual and asexual generations alternate in the life-cycle of the colonial species; stem cells evidently contribute the source of cells in asexual as well as sexual reproduction.

Embryo cryopreservation and in vitro culture of preimplantation embryos in Campbell's hamster (Phodopus campbelli)

The aims of this study were to compare different protocols of Campbells hamster (Phodopus campbelli) embryos freezing-thawing and to explore the possibilities of their inxa0vitro culture. First, the embryos were flushed from the reproductive ducts 2xa0days post coitum at the two-cell stage and cultured in rat one-cell embryo culture medium (R1ECM) for 48xa0hours. Most (86.7%) of the two-cell embryos developed to blastocysts in R1ECM. Second, the embryos at the two- to eight-cell stages were flushed on the third day post coitum. The eight-cell embryos were frozen in 0.25xa0mL straws according to standard procedures of slow cooling. Ethylene glycol (EG) was used either as a single cryoprotectant or in a mixture with sucrose. The survival of frozen-thawed embryos was assessed by double staining with fluorescein diacetate and propidium iodide. The use of EG as a single cryoprotectant resulted in fewer alive embryos when compared with control (fresh embryos), but combined use of EG and sucrose improved the survival rate after thawing. Furthermore, granulocyte-macrophage colony-stimulating factor rat (2xa0ng/mL) improved the rate of the hamster frozen-thawed embryo development inxa0vitro by increasing the final cell number and alleviating nuclear fragmentation. Our data show the first attempt in freezing and thawing Campbells hamster embryos and report the possibility of successful inxa0vitro culture for this species in R1ECM supplemented with granulocyte-macrophage colony-stimulating factor.

Embryo development and embryo transfer in the European mink (Mustela lutreola), an endangered mustelid species

The European mink is an endangered Mustelidae species and thus requires effective conservation measures, although little is known about reproduction in this species. In particular, preimplantation development has not been studied and, therefore, embryonic development and the growth of embryos was documented in the present study for European mink using light and fluorescent microscopy. Embryos develop in the oviducts and then migrate into the uterus on Day 6 post coitum (p.c.) at the morula stage. Embryos expanded as blastocysts from Day 7 until implantation on Day 12 p.c. Based on these findings, the use of embryo transfer for a conservation programme for the European mink was evaluated. Embryos were flushed from European mink resource females and transferred into the uterine horns of recipient hybrid females (honoriks and nohoriks). These hybrids were obtained by mating European polecat males with European mink females and vice versa. A total of 40 embryos was transferred and 20 live kits were born. The rates of pre- and postnatal survival were 50% and 70%, respectively. Both male and female offspring were lighter at birth in the embryo transfer group compared with naturally born controls, but there was no difference at 3 months of age.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae.

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron-carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50 nm to 2 μm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.

Applying reproductive technologies and genome resource banking to laboratory animals

The Genome Resource Bank (GRB) is a repository of frozen biological material, including semen and embryos. Cryobanking is often used in combination with modern reproductive technologies, such as rederivation, in vitro culture, and embryo transfer. Thirteen mouse and rat strains have been rederived, and 32 are kept in cryostorage at the Institute of Cytology and Genetics, Novosibirsk. Other laboratory animal species have been cryopreserved as well. Embryos of two hamster species (Djungarian and Campbell’s) of the Phodopus genus were cryopreserved, and the viability of thawed embryos was confirmed by their successful development in vitro and in vivo (by transfer to a recipient). The positive effect of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) was demonstrated for both of these Phodopus species. Furthermore, the sperm of Djungarian (Phodopus sungorus) and Campbell’s (Phodopus campbelli) hamsters, domestic cat (Felis catusf),leopard cat (Prionailurus bengalensis euptilurus), and bobcat (Lynx rufus) was frozen and cryopreserved. Double staining by SYBR Green/PI, followed by confocal microscopy, demonstrated that more than 40% of amur cat semen retained their viability after cryopreservation. This is the world’s first reported successful freezing of semen of these wild feline species (Prionailurus bengalensis euptilurus). The article reviews the results and discusses the prospects of using reproductive technologies for the conservation of laboratory animal species.

Germline-Restricted Chromosome (GRC) is Widespread among Songbirds

The genome of flying birds, the smallest among amniotes, reflects overweight of the extensive DNA loss over the unrestricted proliferation of selfish genetic elements, resulted in a shortage of repeated sequences and lack of B-chromosomes. The only exception of this rule has been described in zebra finch, which possesses a large germ-line restricted chromosome (GRC), transmitted via oocytes, eliminated from male postmeiotic cells and absent in somatic cell. It is considered as a rarity and its origin, content and function remain unclear. We discovered that all songbirds possess GRC: in various size and genetic content it is present in all fifteen songbird species investigated and absent from germ-line genomes of all eight species of other bird orders examined. Our data based on fluorescent in situ hybridization of DNA probes derived from GRCs of four different Passeri species and their sequencing indicate that the GRCs show low homology between avian species. They contain fragments of the somatic genomes, which include various unique and repetitive sequences. We propose that the GRC has formed in the common ancestor of the extant songbirds and undergone subsequent divergence. GRC presence in the germ line of every songbird studied indicate that it could contain genetic element(s) indispensable for gametogenesis, which are yet to be discovered.

Sperm cryopreservation in the Far-Eastern wildcat (Prionailurus bengalensis euptilurus)

The Far-Eastern wildcat (Prionailurus bengalensis euptilurus) is a rare and poorly investigated nondomestic felid species. An attempt of freezing and cryopreserving Far-Eastern wildcat spermatozoa in CaniPlus Freeze (CPF) medium is reported. Sperm was collected by electroejaculation from five adult Far-Eastern wildcat captive-born males. Epididymal spermatozoa from five adult randomly bred domestic cat males were used as a reference. The viability of frozen-thawed spermatozoa evaluated by double staining with SYBR Green I and PI followed by the subsequent confocal laser scanning microscopy (CLSM) was 38.2%xa0±xa03.0% for the domestic cat and 38.0%xa0±xa010.2% for the Far-Eastern wildcat. The motility of frozen-thawed spermatozoa was 30.8%xa0±xa09.8% for the domestic cat and 33.7%xa0±xa015.1% for the Far-Eastern wildcat. Sperm morphology was assessed by light microscopy. The total percentage of normal spermatozoa after freezing and thawing was 51.9xa0±xa05.9 for the domestic cat and 55.0%xa0±xa06.4% for the Far-Eastern wildcat. Defects of flagella were the most frequently observed abnormalities in both species (32.2%xa0±xa04.8% and 30.8%xa0±xa04.4% of all reported anomalies for the domestic cat and Far-Eastern wildcat, respectively). Domestic cat epididymal and Far-Eastern ejaculatory spermatozoa fertilized in vitro-matured oocytes of the domestic cat (30.0%xa0±xa05.5% and 35.5%xa0±xa015.0%, respectively). Taken together, these results suggest that the freezing of Far-Eastern wildcat spermatozoa with CPF medium is a suitable method for Felidae cryopreservation.

Mitochondria structural reorganization during mouse embryonic stem cell derivation

Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5xa0days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23–36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~u200919% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.

Chromosome Synapsis and Recombination in Male-Sterile and Female-Fertile Interspecies Hybrids of the Dwarf Hamsters (Phodopus, Cricetidae)

Hybrid sterility is an important step in the speciation process. Hybrids between dwarf hamsters Phodopus sungorus and P. campbelli provide a good model for studies in cytological and genetic mechanisms of hybrid sterility. Previous studies in hybrids detected multiple abnormalities of spermatogenesis and a high frequency of dissociation between the X and Y chromosomes at the meiotic prophase. In this study, we found that the autosomes of the hybrid males and females underwent paring and recombination as normally as their parental forms did. The male hybrids showed a significantly higher frequency of asynapsis and recombination failure between the heterochromatic arms of the X and Y chromosomes than the males of the parental species. Female hybrids as well as the females of the parental species demonstrated a high incidence of centromere misalignment at the XX bivalent and partial asynapsis of the ends of its heterochromatic arms. In all three karyotypes, recombination was completely suppressed in the heterochromatic arm of the X chromosome, where the pseudoautosomal region is located. We propose that this recombination pattern speeds up divergence of the X- and Y-linked pseudoautosomal regions between the parental species and results in their incompatibility in the male hybrids.