Assessment of Embryo-Induced Transcriptomic Changes in Hamster Uterus Using RNA-Seq.

BACKGROUND/AIMS: The mouse is widely used as an animal model for studying human embryo implantation. However, the mouse is unique in that both ovarian progesterone and estrogen are critical to implantation, whereas in the majority of species (e.g. human and hamster) implantation can occur in the presence of progesterone alone. METHODS: In this study, we analyzed embryo-induced transcriptomic changes in the hamster uterus during embryo implantation by using RNA-seq. Differentially expressed genes were characterized by bioinformatic analysis. RESULTS: We identified a total of 781 differentially expressed genes, of which 367 genes were up-regulated and 414 genes were down-regulated at the implantation site compared to the inter-implantation site. Functional clustering and gene network analysis highlighted the cell cycle process in uterus upon embryo implantation. By examining of the promoter regions of differentially expressed genes, we identified 7 causal transcription factors. Additionally, through connectivity map (CMap) analysis, multiple compounds were identified to have potential anti-implantation effects due to their ability to reverse embryo-induced transcriptomic changes. CONCLUSION: Our study provides a valuable resource for in-depth understanding of the mechanism underlying embryo implantation.

Cell cycle-dependent dynamics of cytoskeleton involving mitochondrial redistribution in hamster embryos.

Mitochondria-cytoskeleton interactions were studied in the hamster embryos during interphase and M phase of the cell cycle. Two-cell embryos were cultured for 1 h with nocodazole, cytochalasin D or in a combination of both inhibitors and then centrifuged at 10,000 × g for 2 min. The control embryos were only centrifuged with no inhibitor treatment. Centrifuged embryos were fluorescently stained to examine the distribution of active mitochondria and nuclear configuration. In the control 2-cell embryos, most mitochondria were accumulated at the perinuclear region with some at the cell cortex. Neither each inhibitor nor centrifugation did affect the distribution of mitochondria in interphase blastomeres. However, mitochondria were spun down towards the centrifugal pole in 71% (n = 41) of the interphase blastomeres treated with centrifugation following a combination of nocodazole plus cytochalasin D, suggesting that both microtubules and microfilaments may involve in mitochondrial redistribution during interphase of the cell cycle. In contrast, when M-phase blastomeres were treated with all drug treatments applied, including cytochalasin D, mitochondria had been usually dislocated in a unipolar cluster, suggesting that microfilaments, not microtubules, may involve in the mitochondrial redistribution during M phase of the cell cycle. The data indicate that microfilaments function in mitochondrial redistribution regardless of the stages of the cell cycle and that microtubules may strongly associate with mitochondria during the interphase but dissociate from them during the M phase.

Repopulation of the postmitotic nucleolus by preformed RNA.

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04-0 08 microg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04-0 08 microg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis

Quantitative in vitro transformation of Syrian golden hamster embryo cells with the use of frozen stored cells.

Quantitative transformation by carcinogens of Syrian golden hamster embryo cells from primary, secondary, or tertiary cultures could be obtained from cell pools frozen prior to any culturing with the same efficiency as with fresh cells. Cells retained the ability to activate a wide range of chemical carcinogens when the noncultured hamster cells were cooled at a controlled rate and when the established procedures for culturing cells were followed. The number of experiments that could be done with cells derived from frozen hamster pools could be increased by substitution of a hamster cell line for the feeder layer. The response to chemical carcinogens of different classes including carcinogenic hydrocarbons, aromatic amine derivatives, metal complexes, aflatoxin B1, N-methyl-N'-nitro-N-nitrosoguanidine, and UV irradiation was similar to that reported with fresh cells. Transformation rate increased with increasing carcinogen concentration. The factors important for the breeding of healthy animals and for selection of those appropriate for obtaining reproducible transformation experiments were enumerated.

In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard assessment.

The globin gene expression program in the hamster embryo.

Molecular mechanisms involved in control of globin gene expression are a prominent target in current basic biologic research. A better understanding of these mechanisms might also impinge on a clinical goal: amelioration of the human hemoglobinopathies. Recent reports have established the coexistence of embryonic and adult globins in rodent yolk-sac erythroid cells, raising the possibility that globin ontogeny takes place in these cells. The present study was undertaken to define the extent of this putative ontogenic process. We measured daily rates of synthesis of individual globins in hamster yolk-sac erythroid cells from the earliest day in gestation that these cells are available (day 7) until the day they cease to replicate (day 13). Converted to a per-cell basis, the rates demonstrate an ontogenic progression in globin synthesis, from embryonic globins to adult globins, that encompasses nearly entirely the total globin ontogeny of this mammal. Synthesis of adult alpha globin is already detectable on day 7, whereas synthesis of the two adult beta globins does not appear until day 9. Synthesis of embryonic y globin stands in contrast to that of the other two embryonic globins (x and z), rising as they fall, a phenomenon reminiscent of the gamma globin of primates and certain ruminants. This physiologic primitive erythroid cell appears to be an unusually appropriate target for studies directed at globin ontogeny.

Duration of retinogenesis: its relationship to retinal organization in two cricetine rodents.

The Mongolian gerbil (Meriones unguiculatus) has a prolonged period of development relative to other muroid rodents. We have explored the consequences of this relatively long period of maturation on retinal cell number and topography by comparing the duration and topography of neurogenesis in the gerbil retina with that of a closely related species which develops rapidly, the Syrian hamster (Mesocricetus auratus) (Sengelaub et al.: J. Comp. Neurol. 246:527-543, 1986). An analysis of thymidine-labeled retinas indicate that cells destined for the gerbil retinal ganglion cell layer are generated for at least 12 embryonic days, twice the duration in the hamster. The period of cell loss in the gerbil retinal ganglion cell layer extends for at least 14 postnatal days, more than twice as long as in the hamster. The gerbil retina is generated in a center-to-periphery gradient for both retinal ganglion cells and displaced amacrine cells, while no such gradients are evident in the hamster retina. We conclude that the longer developmental period of the gerbil is associated with 1) a longer period of neurogenesis resulting in greater retinal cell number, 2) the expression of spatial gradients in neurogenesis, and 3) a larger eye at maturity. The last two factors, in part, may be related to the development of a highly differentiated area centralis and visual streak in the retina of this rodent. Unrelated to duration of growth, early differences in retinal shape between these two species contributes to the development of retinal topography. The gerbil, but not the hamster retina, is initially asymmetric, longer in its nasotemporal than its dorsoventral dimension. The gerbil retina then grows asymmetrically, producing a spherical retina, and coincident in time, a nasotemporally extended visual streak.

Molecular tunnels and boundaries for growing axons in the anterior commissure of hamster embryos.

We have analyzed the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), laminin (LN), tenascin (TN), and glial fibrillary acidic protein (GFAP) along the anterior commissure (AC) of hamster embryos (n=175; from embryonic day (E)12 to E16). Frozen sections were cut at different planes from embryonic brains between E12 and E16, treated for immunohistochemistry, and observed under epifluorescence microscopy. During the pre-crossing stage (E12-E13), CSPG was expressed as a sagittal stratum between the interhemispheric fissure and the prospective AC region. TN appeared rostral to the third ventricle and along the medial subventricular zone of the lateral ventricles. LN and FN both presented a faint expression, and GFAP was not detected. Although AC axons started crossing the midline region (E13.5-E14), CSPG, FN, LN, and, much less intensely, GFAP circumscribed the AC bundle, forming a tunnel through which AC fibers elongate. TN was no longer seen at the midplane but remained visible laterally. During the post-crossing stage (E14.5-E16), CSPG and TN were no longer seen at the midline, although both could be observed between the AC limbs, seeming to form boundaries for AC lateral growth. LN and FN were then absent near the AC bundle. During this late stage, GFAP expression became most intense, forming a distinct tunnel around the AC. We have shown that the expression of extracellular matrix molecules and GFAP follow a time- and space-regulated course related to AC development, plausibly representing influential factors for growth and guidance of commissural fibers.

Ageing of hamster embryo fibroblasts as the result of both differentiation and stochastic mechanisms.

Fibroblasts from hamster embryos were serially cultivated in vitro and their evolution followed from a morphological, physiological and biochemical point of view. After an exponential growth for about 20 passages, cells entered the ageing phase which ended up after 29-34 passages. From our observations, it seems that the arrest of growth results from two different phenomena: first, the typically fibroblastic cells may undergo a stochastic ageing process; second, some of these cells evolve into a terminal differentiation process, characterized by a different non-fibroblastic phenotype.

Microtubules and microfilaments in ageing hamster embryo fibroblasts in vitro.

Microtubules and microfilaments were investigated in hamster lung fibroblasts, during their in vitro life-span. These cells show a senescence process characterized by a drastic phenotypic change, resulting in two phenotypes: the type 1 cells, characteristic of young cultures and the type 2 cells appearing progressively with culture passages. Microtubules and microfilaments were observed at the TEM and also visualized by the unlabelled peroxidase-anti-peroxidase method. Moreover, the susceptibility of microtubules to nocodazole was tested in type 1 and 2 cells. We could not provide evidence for a different susceptibility to the drug. However the depolymerization wave occurred centripetally in type 1 cells whilst centrifugally in type 2 cells. These observations are discussed in relationship with the early arrest of division growth of the type 2 differentiated cells.

Arsenic as a teratogenic agent.

Sodium arsenate induces developmental malformations in a variety of experimental animals. In the golden hamster, the intravenous (or intraperitoneal) administration of 20 mg/kg of sodium arsenate during day 8 to 9 of gestation induces a rather specific spectrum of congenital malformations. This period corresponds to the period of very rapid differentiation and major organogenesis in this animal. The spectrum of defects produced by arsenate in the hamster includes exencephaly, encephaloceles, skeletal defects, and malformations of the genito-urinary system. This teratogenic effect can be significantly reduced by the simultaneous administration of selenium. Recent studies in this laboratory have demonstrated the permeability of the placenta to 74As during the early critical stages of embryogenesis and the distribution of this isotope in maternal, placental and embryonic tissues. We have also recently demonstrated the marked potentiation of the teratogenic effect of sodium arsenate by subjecting the mothers to short periods of hyperthermia immediately following the administration of subteratogenic or minimal teratogenic levels of arsenate.

Use of brainstem flat-mounts for visualizing DiI-filled axons in the developing rodent visual system.

The lipophilic carbocyanine fluorescent label DiI was injected in one eye of aldehyde-fixed embryonic or postnatal hamsters and the brains were examined using flat-mounts of the chiasm region, of the lateral surface of the brainstem, or of the midbrain tectum. Single axons could be discerned within the optic nerves and along the optic tract. Many fibers were tipped by growth cones, ending at various levels of the brainstem. Fine details of retinofugal axon morphology, including varicosities, branch-points and filopodial extensions on growth cones were visible in the flat-mounts. Such preparations allow a high-resolution view of labeled axons which course near the surface of the brain. It is possible, with this method, to simultaneously examine the morphogenesis of multiple collateral arbors on single fibers which project to more than one terminal zone.

Appearance of melatonin receptors during embryonic life in Siberian hamsters (Phodopus sungorous).

Maternal melatonin readily crosses the placenta to provide the fetus with time-of-day and day length information. To determine if melatonin could have a broader role during development than is currently recognized, melatonin receptor expression was examined in somatic sites during Siberian hamster embryogenesis. Using 125I-labeled-2-iodomelatonin ([125I]MEL), melatonin receptor expression was examined in whole fetuses by in vitro autoradiography. [125I]MEL binding sites were first apparent at gestational day (GD) 10 over the primitive oral pharynx. From GD 12 to 14, binding was present over the nasal pharynx, Rathke's pouch, caudal arteries, and over the thyroid gland during its migration along the thyroglossal duct. By GD 16, Rathke's pouch had differentiated into the pituitary gland, which continued to express specific [125I]MEL binding until birth. From GD 16 until birth, binding was no longer detectable over the thyroid gland, but persisted over the nasal epithelium. At all ages, binding sites exhibited high affinity for [125I]MEL and appeared to be coupled with guanosine nucleotide-binding proteins. These data suggest that melatonin receptors are expressed in several somatic sites, including Rathke's pouch and the thyroid gland, during fetal development.

Prenatal development of the microphthalmic eye in the golden hamster.

Prenatal development of the eye in a microphthalmic hamster strain ("anophthalmic white") is compared with established normal developmental periods. The mutant eye primordium is first distinguished at an average of ten gestational days (Period 6) by an incompletely invaginated optic cup, uniformly pseudostratified outer neuroepithelial layer and widely separated margins of the optic fissure. The outer layer of the mutant cup subsequently becomes abnormally thickened, especially posteriorly and midventrally, and, except in a few eyes with localized imperfect fusion, the optic fissure is unfused at twelve days (Period 9), by which time fusion is normally complete. At 13 to 15 days (Period 10-11) the fissure is unfused or irregularly fused in regions of variable location and extent. The occurrence of fissure fusion with concomitant loss of continuity between inner and outer epithelial layers is generally restricted to expanded anterior regions in 14-16 day (Periods 11-12) eyes. The presence of presumptive neural retina in the outer layer of the cup characterizes the mutant eye; and to varying degrees, in day 13-16 eyes, the presumptive neural retina (1) provides persistent continuity between the two cup layers, (2) forms both fused and unfused margins of the optic fissure, and (3) extends into an outer position of the optic cup. As early as 13 days (Period 10), nerve fibers are present in the outer layer of the cup, and by the last prenatal and first postnatal days (Period 12), ectopic nerve fiber bundles are widely distributed.

Stages in the prenatal development of the Chinese hamster (Cricetulus griseus).

A system of staging embryos is described for the Chinese hamster (Cricetulus griseus). This system of staging, based on Streeter's developmental horizons in human embryos, comprises three sets of criteria: 1) data on postconceptional age, size and number of somites, 2) external characteristics and 3) internal characteristics. A comparison has been made with data in mouse (Theiler, 1972) as well as in primates. It seems that the order of organogenesis, i.e. the sequence in which individual organs are formed, is basically similar in all mammals stuied so far.

Neurogenesis in the basal forebrain in the Chinese hamster (cricetulus griseus). II. Site of neuron origin: morphogenesis of the ventricular ridges.

In the present study the morphogenesis of the ventricular ridges, i.e. the site of origin for neurons in the basal ganglia and various related basal forebrain structures, has been studied in the Chinese hamster with the aid of three-dimensional and graphical reconstructions. The first ridge appears at developmental stage 14 (E 12 1/2). It originates at the level of the torus hemisphaericus, thereby obscuring the basal part of the telodiencephalic boundary. Later on this ridge passes into the medial ventricular ridge. Subsequently, the lateral ventricular ridge arises at stage 16 (E 13 1/2). Initially, both ridges are completely separated by the sulcus subpallii intermedius. During further development, however, this limiting groove fades away, a process starting caudally and gradually proceeding in the rostral direction. Eventually, this process results in the formation of one single ventricular eminence at the second fetal stage (E 18). In the adult stage the ventricular eminence curves around the cerebral stem area. Thickening of the telencephalic walls and local coarctations have considerably reduced the lumen of the lateral ventricle. The preoptic region in the adult must be considered as a derivative of the diencephalic part of the medial ventricular ridge.

Effects of ovariectomy and ageing on the structure and ultrastructure of the female Syrian hamster Harderian gland: a stereological analysis.

The effects of ovariectomy and ageing on the structure and ultrastructure of the Syrian hamster Harderian gland were investigated by techniques of quantitative stereology. Tissues were obtained from intact 6-month-old, sham-operated 6-month-old, ovariectomized 6-month-old, intact 18-month-old and ovariectomized 18-month-old female hamsters. Glands from both ovariectomized and aged hamsters showed comparable qualitative and quantitative characteristics. They showed histological alterations that included thinning of the tubule walls, lowering of luminal porphyrins, invasion of lumina by neutrophils and the occurrence of interstitial porphyrins. Glands from both ovariectomized and aged hamsters showed statistically significant differences from control animals in relation to numerical density and cellular size. Finally, quantitative studies with the electron microscope revealed significant decreases in the volume densities of the cytoplasmic organelles concerned with secretion. These results support the hypotheses that the secretory activity of the female hamster Harderian gland is influenced, directly or indirectly, by ovarian hormones, and that many of the age-related modifications of the Harderian gland reflect alterations in ovarian function.

Neurogenesis in the basal forebrain of the Chinese hamster (Cricetulus griseus). I. Time of neuron origin.

The time of neuron origin has been determined in the basal ganglia and related basal forebrain structures of the Chinese hamster with the aid of 3H-thymidine autoradiography. Large-celled structures like the globus pallidus, nucleus of the horizontal limb of the diagonal band of Broca as well as large cells in the rostral part of the substantia innominata, in the caudate-putamen-complex and in the olfactory tubercle arise early (E12--E16), whereas medium-sized and small cells in the basal forebrain have a persistent origin over a much longer period. Neuron formation in the basal forebrain persists decrementally until P4. A clear caudorostral spatiotemporal gradient as well as a distinct 'outside-in' gradient have been observed in the caudate-putamen-complex. Medium-sized neurons in the neostriatum and in the nucleus accumbens, generated simultaneously, are usually arranged in scattered clusters. The present data on time of neuron origin strongly support other evidence which points to the conclusion that the nucleus accumbens can be considered as a ventromedial extension of the caudate-putamen-complex.

The embryological development and cytodifferentiation of the pars distalis of the golden hamster (Mesocricetus auratus). A light and electron microscopic study.

The embryological development of cytodifferentiation of the hamster pars distalis was investigated using light and electron microscope techniques in order to obtain basic information for comparison with pituitary development in other mammalian species. The normal chronological events in the development of the hamster pars distalis closely paralleled the pituitary organogenesis of other laboratory rodents. Rathke's pouch formed and touched the infundibulum at 8 1/2 days of gestation and separated from the stomodeum 3 days later. Penetration of vascular elements from the developing hypophysial portal system into the pars distalis occurred at 12 1/2 days gestation. This was also the first day that small secretory granules were seen in any of the parenchymal cells. Further cytodifferentiation during the following prenatal, and first few postnatal days of life revealed granulated cells which, in most cases, could not be identified using morphological criteria or granule size as may be done in the adult. An orderly sequence of inductive and morphological events appears to take place in the developing hamster adenohypophysis paralleling similar events observed in other animals.

Chromosomal aberrations and morphological transformation in hamster embryonic cells treated with potassium dichromate in vitro.

The addition of K2Cr2O7, at concentrations ranging from 0.1 to 0.5 microng/ml, to hamster total embryonic cells for 24 h, resulted in consistent and drastic chromosomal aberrations including gaps, breaks and exchanges. The above effect, however, was reduced successfully by the addition of a reducing agent, Na2SO3. Among other chromium compounds examined, divalent and trivalent chromium salts were ineffective on chromosome morphology even at a concentration of 3.5 microng/ml as chromium, whereas a hexavalent compound, CrO3, was highly effective. K2Cr2O7 also enhanced the morphological transformation rate in a short-term colony assay, in whicy hamster embryonic cells (1x10(4) cells/60-mm dish) were treated and the morphology was observed 8 to 10 days after the treatment.