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.

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.

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.

Full-term development of hamster embryos produced by injection of round spermatids into oocytes.

The golden hamster is a mammal in which microinjection of round spermatids into oocytes (ROSI) was first attempted. However, no live ROSI offspring have ever been obtained in this species. This is the first report of live hamster offspring obtained by round spermatid injection. Over 90% of oocytes, injected with round spermatids, were activated without any additional stimulation. The proportion of the oocytes that were fertilized normally and that developed to morulae and blastocysts was higher when the plasma membranes of the spermatids were broken before injection, as compared with when the membranes were left intact. Five percent of 57 ROSI morulae/blastocysts developed into live offspring after transfer to foster mothers.

Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production.

The effects of water-soluble vitamins, singly or in combinations, on development of hamster 1-cell embryos were examined in a protein-free, chemically defined culture medium, HECM-6. Pantothenate significantly stimulated blastocyst development compared to the vitamin-free control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. When combinations of vitamins were tested, embryo development was either unchanged or significantly greater than in the control, but never significantly greater than development with pantothenate alone. A dose response to pantothenate indicated that 3 micromol/l was the optimum concentration. After embryo transfer, the percentage of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate (now designated HECM-9) was 24%, significantly higher than the 11% recovered from 100 1-cell embryos cultured in HECM-6 alone. This is the first report to show a stimulatory effect of a single vitamin on in-vitro development of preimplantation embryos in any mammalian species.

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.

Quantitative and topographic analysis of sulphur in the zona pellucida from mouse, hamster and rabbit oocytes

Mediante la técnica del microanálisis de rayos X, se investigó la cantidad y distribución del azufre en la zona pelúcida de los ovocitos de ratón, hamster y conejo. La cantidad de azufre varía entre 2853 y 10178 mmol/Kg de peso seco. La cantidad más alta de azufre se encontró en los ovocitos de hamster y la más baja en los ovocitos de conejo; siendo la cantidad encontrada en los ovocitos de ratón, intermedia entre ambas. Se detectó una disminución gradual del azufre desde la región interna hacia la región externa de la zona pelúcida, en los ovocitos de ratón y conejo. Esto es coincidente con las asimetrías morfológica y/o bioquímica descritas para la zona pelúcida de estas especies. Aunque se han descrito asimetrías bioquímica y funcional en la zona pelúcida de los ovocitos de hamster, en el presente análisis no se detectó una distribución asimétrica del azufre. Sin embargo, resultó evidente una estratificación de este elemento en la zona pelúcida de los ovocitos de hamster

Expression of the human beta 2-adrenoceptor in NCB20 cells results in agonist activation of adenylyl cyclase and agonist-mediated selective down-regulation of Gs alpha.

Murine neuroblastoma x embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human beta 2-adrenoceptor under the control of a beta-actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the beta-adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild-type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein alpha subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane-associated levels of Gs alpha in membranes of clone L9 cells and a 50% reduction in Gs alpha levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gs alpha in wild-type NCB20 cells, and such treatment had no effect on the levels of other G protein alpha subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half-maximal loss of Gs alpha in clone D1 was achieved within 1-2 h of addition of agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Entrainment of the fetal hamster circadian pacemaker by prenatal injections of the dopamine agonist SKF 38393.

Prenatal treatment with the D1-dopamine receptor agonist SKF 38393 or cocaine induces expression of the immediate-early gene c-fos in the fetal rat suprachiasmatic nucleus (SCN) (Weaver et al., 1992). Because the induction of c-fos gene expression in the SCN has been implicated in the entrainment of circadian rhythms by light in mature animals, the present study investigated whether prenatal dopaminergic activation entrains the fetal circadian pacemaker. Injections of SKF 38393 (8 mg/kg) were given to pregnant, SCN-lesioned hamsters during the last 5 d of gestation and the phases of the offspring's wheel-running activity rhythms were measured on postnatal day 20. Pregnant hamsters were each given two injections/day 12 hr apart, but only one of the injections each day contained SKF 38393. One group of hamsters received the drug at 0800 hr while another group received the drug at 2000 hr. The offspring from these treatment groups showed average phases that differed by 11.3 hr, demonstrating that prenatal SKF 38393 set the phase of the offspring's circadian rhythms. These results suggest that the fetal circadian pacemaker can be entrained by dopaminergic activation. In situ hybridization using cRNA probes demonstrated that a single injection of SKF 38393 on the last day of gestation induced c-fos gene expression in the fetal hamster SCN and that mRNA for the D1-dopamine receptor was present in the SCN at that time. It is possible that maternal entrainment of the fetal circadian pacemaker, which normally occurs during development, is mediated by dopaminergic activation within the fetal hypothalamus.

Topographic organization in the anterior commissure of developing hamsters.

The present study has examined the existence of a topographic organization in the anterior commissure (AC) of developing hamsters. Fluorescent carbocyanine crystals (DiI and/or DiA) were implanted into different rostrocaudal and dorsoventral sectors of the paleocortex of hamsters ranging in age from E15 to P10 (E16 = P1 = date of birth). The cerebral hemispheres of each brain were cut horizontally and sagittally, respectively, and the sections were observed under a fluorescence microscope coupled to a computerized reconstruction system. A distinct topographic organization of AC fibers was observed along the rostrocaudal axis starting at E15, and continued unchanged thereafter. These results support the hypothesis that the orderly pattern of AC fibers is achieved by active positioning during the first days after crossing the midplane rather than by a regressive sculpting from an initially disorganized pattern.

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.

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.

Genesis of hadacidin-induced cleft palate in hamster: morphogenesis, electron microscopy, and determination of DNA synthesis, cAMP, and enzyme acid phosphatase.

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.

Cytoskeleton of the mouse egg and embryo: reorganization of planar elements.

Examination of detergent-extracted mouse eggs and embryos reveals the existence of two cytoskeletal networks. One network is the typical thin filament network observed in somatic cells while the other is composed of large planar elements. These latter cytoskeletal structures, with individual widths of 60.0 +/- 6.8 nm, alter their spatial organization in a developmental stage-specific manner. The planar elements are composed of filaments with a diameter of 10 nm aligned side-by-side with these filaments exhibiting a linear periodicity of 20.0 +/- 1.6 nm. A biochemical fraction containing components of the planar elements has been prepared from different stages of development and disappearance of prominent polypeptides from this fraction correlates with the altered spatial organization of the planar elements. Ultrastructure and biochemistry of cytoskeletal planar elements in eggs and embryos of the mouse are comparable with cytoskeletal sheets of Syrian hamster eggs and embryos, suggesting these cytoskeletal components may have a functional role in mammalian embryogenesis. Because such structures have not been identified in eggs or embryos of species other than mammals, their function may be unique to mammalian embryogenesis.

Nuclear localization of 68 kDa calmodulin-binding protein is associated with the onset of DNA replication.

In Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin-binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin-binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin-binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin-binding protein, along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin-binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.

Environmental variables influencing in vitro development of hamster 2-cell embryos to the blastocyst stage.

This study is a systematic analysis of environmental variables influencing development of 2-cell hamster embryos to the blastocyst stage in vitro. Experiments were done using a chemically defined (protein-free) culture medium (HECM-2). Physicochemical variables examined were temperature, the concentrations of CO2, HCO3-, Ca2+, Mg2+, K+, and O2, the presence of a silicone oil overlay, and osmotic pressure. The optimal temperature for development in vitro was 37.5 degrees C; lower temperatures were inhibitory. There was no significant effect on blastocyst development of alterations in the concentrations of NaHCO3, CaCl2, MgCl2, and KCl, or in the ratios of Ca2+:Mg2+ and Na+:K+, over the ranges tested. Development to the blastocyst stage was significantly stimulated by increased CO2 concentrations (7.5% and 10%), by reduced O2 concentrations (10% and 5%), and by the presence of silicone oil overlying the culture medium. Reduction of blastocyst development in the absence of an oil overlay was not caused by increased osmotic pressure. Cleavage stage embryos were not affected by osmolalities ranging from 250 to 350 mOsmols, but blastocyst development was inhibited at greater than or equal to 325 mOsmols. Under optimized conditions (37.5 degrees C, 10% CO2, 25 mM HCO3-, 2.0 mM Ca2+, 0.5 mM Mg2+, 3.0 mM K+, 10% O2, 250-300 mOsmols, with silicone oil overlay), 51-57% of 2-cell hamster embryos developed to the blastocyst stage. This represents a significant improvement over previous "standard" culture conditions, which supported development of 26% blastocysts from 2-cell hamster embryos. The culture system described here provides an adequate baseline response to support detailed investigations into the regulation of embryo development in the hamster.

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.

Reception of photoperiodic information by fetal Siberian hamsters: role of the mother's pineal gland.

The rate of reproductive development in juvenile male Siberian hamsters is strongly influenced by daylength (photoperiod). Recent studies indicate that reception of photoperiodic cues begins during fetal life. The present experiments yielded a further demonstration that developing male Siberian hamsters receive information about the photoperiod to which their mother is exposed during pregnancy. The possibility that photoperiodic information is transmitted from mother to young after birth was investigated by cross-fostering young gestated on 12L and 16L to mothers from the other photoperiod. Litters were cross-fostered on the day of birth and then were transferred, along with their foster mothers, to 14L. We found no influence of the mother after birth, indicating that transmission of photoperiodic information from mother to young must occur during gestation. To determine if the pineal gland of the mother is required for this response, adult females were pinealectomized or sham-operated and paired with intact males in 12L, 14L, or 16L. After parturition parents and offspring were exposed to 14L. The influence of prenatal photoperiod on postnatal testicular development in 14L was blocked by pinealectomy of the mother. Postnatal testicular development was retarded in offspring that experienced a photoperiod transfer from either 15L to 14L or 8L to 12L at birth. In contrast, the inhibitory effect of a transfer from 16L to 14L at birth was abolished when juvenile males were exposed to a single long photoperiod (16.3 h light) at age 17-21 days and then were returned to 14L.

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.