Embryonic Development In Rat Research Articles

Study of the toxicity of topiramate on embryonic development in rats

目的 研究妥泰(topiramate,TPM)对大鼠胚胎发育的毒性. 方法将妊娠SD大鼠随机分为五组.实验组于妊娠6～15 d 经口灌胃给予TPM 40 mg/kg,TPM 80 mg/kg和TPM 40 mg/kg+丙戊酸钠(VPA)300 mg/kg.阴性对照组同期经口灌胃给以等量的蒸馏水.阳性对照组于妊娠第11天一次性腹腔注射环磷酰胺(CP)10 mg/kg.于妊娠第20天处死孕鼠,观察分析活胎、死胎和吸收胎数、仔鼠身体外形畸形及有关发育指标. 结果实验组的TPM 40 mg/kg组、80 mg/kg组和TPM 40 mg/kg+ VPA 300 mg/kg组母鼠体重增长分别为 (89.6±16.5)、(74.8±10.7)、(91.5±20.3)g,显著低于阴性对照组(109.5±14.2)g( t值分别为2.9、 6.4、2.3,P值分别为<0.01,<0.001和<0.05).仔鼠的平均体重、身长、尾长指标明显降低;上述 TPM三组的死胎率和吸收胎率增加,活胎数减少,分别为96 %、88 %、86 %,阴性对照组为97 %,植入数中活胎和死胎+吸收胎间的构成比,在大剂量及与VPA联合用药组差异有非常显著性(χ2值分别为7.7、9.95,P均<0.01).仔鼠脐疝、短尾增多. 结论 TPM在小剂量时对胚胎的发育影响较小,而大剂量及与丙戊酸钠联合用药时影响较大。

Effects of 4-hydroxyandrostenedione and hyperstimulation with pregnant mare serum gonadotrophin on early embryonic development in rats

A possible role of high oestradiol levels in mediating the adverse effects of hyperstimulation with pregnant mare serum gonadotrophin (PMSG) on early embryonic development in the rat was investigated using an aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA), to inhibit endogenous oestradiol production. Three experiments were conducted in this study. In the first, varying doses of 4-OHA were administered either concurrently with human chorionic gonadotropin (hCG) to pro-oestrus female rats hyperstimulated at early di-oestrus stage with 20 IU PMSG or alone into nonhyperstimulated pro-oestrus females. At high doses of 1000, 2000, or 5000 µg/rat, 4-OHA substantially improved the survival of embryos in hyperstimulated females, while low doses of 100 and 500 µg/rat were ineffective. The protective effect of 4-OHA on embryo count was optimum at 2000 µg. When administered alone, only the highest dose of 5000 µg/rat 4-OHA increased embryo count. In the second experiment, higher doses of PMSG were studied (30 or 40 IU), with or without 5000 µg/rat 4-OHA given at the time of hCG injection. PMSG proved to be more detrimental with increasing dose, and 5000 µg/rat 4-OHA was able to rescue embryos from death in the 30, but not 40, PMSG group. In the third experiment, the influence of the timing of 4-OHA treatment on its ability to improve the embryo count in hyperstimulated females was examined by introducing 4-OHA 24 h earlier, rather than at the time of hCG treatment. The results showed the importance of timing of 4-OHA administration, as 5000 µg/rat 4-OHA was able to restore embryo survival in the 40 PMSG hyperstimulated group only when it was administered 24 h before hCG injection. Together, these results highlighted that 4-OHA, when administered at the appropriate time and dose, could reverse the negative effects of hyperstimulation from PMSG on early embryonic development. This may be due to its potent aromatase inhibiting properties that lead to the suppression of oestrogen production, thereby alleviating the supraphysiological level of oestradiol, which is typically present in PMSG-treated females. Interestingly, 4-OHA treatment on its own was able to positively influence embryo count when given at a high dose of 5000 µg/rat, and this may be associated with its weak androgenic properties. In conclusion, this study supports the hypothesis that excessive oestradiol is responsible for the negative effects of hyperstimulation with PMSG on early embryonic development.Key words: 4-hydroxyandrostenedione, embryonic development, PMSG, rat.

Kalirin, a GDP/GTP exchange factor of the Dbl family, is localized to nerve, muscle, and endocrine tissue during embryonic rat development.

Kalirin, a homologue of trio and UNC-73, has been previously demonstrated to cause cytoskeletal rearrangements, enhanced outgrowth of neuritic processes, and altered secretion. In the adult rat, kalirin is specifically localized to the central nervous system, with the main adult isoform, kalirin-7, concentrated in neuronal postsynaptic densities. In this study we examined the expression of kalirin in rat tissue from embryonic Day 10 (E10) through E18, using an antibody that detects all known kalirin isoforms. Kalirin expression in the embryo was more widespread than in the adult, with localization of kalirin protein to both neuronal and non-neuronal tissue, such as muscle, lung, intestinal epithelium, and pancreas. In neurons, kalirin was localized both in cell bodies and axon processes; in muscle tissue, kalirin was highly localized to migrating myogenic cells and at muscle attachment sites. Western blotting analysis indicated that kalirin-7, the major adult isoform, was a minor component of embryonic kalirin; the main isoform expressed in the embryo was kalirin-9. This is the first identification of kalirin expression in embryonic tissue and the first demonstration of non-neuronal expression of kalirin. (J Histochem Cytochem 49:833-844, 2001)

Effects of 4-hydroxyandrostenedione and hyperstimulation with pregnant mare serum gonadotrophin on early embryonic development in rats

A possible role of high oestradiol levels in mediating the adverse effects of hyperstimulation with pregnant mare serum gonadotrophin (PMSG) on early embryonic development in the rat was investigated using an aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA), to inhibit endogenous oestradiol production. Three experiments were conducted in this study. In the first, varying doses of 4-OHA were administered either concurrently with human chorionic gonadotropin (hCG) to pro-oestrus female rats hyperstimulated at early di-oestrus stage with 20 IU PMSG or alone into nonhyperstimulated pro-oestrus females. At high doses of 1000, 2000, or 5000 microg/rat, 4-OHA substantially improved the survival of embryos in hyperstimulated females, while low doses of 100 and 500 microg/rat were ineffective. The protective effect of 4-OHA on embryo count was optimum at 2000 microg. When administered alone, only the highest dose of 5000 microg/rat 4-OHA increased embryo count. In the second experiment, higher doses of PMSG were studied (30 or 40 IU), with or without 5000 microg/rat 4-OHA given at the time of hCG injection. PMSG proved to be more detrimental with increasing dose, and 5000 microg/rat 4-OHA was able to rescue embryos from death in the 30, but not 40, PMSG group. In the third experiment, the influence of the timing of 4-OHA treatment on its ability to improve the embryo count in hyperstimulated females was examined by introducing 4-OHA 24 h earlier, rather than at the time of hCG treatment. The results showed the importance of timing of 4-OHA administration, as 5000 microg/rat 4-OHA was able to restore embryo survival in the 40 PMSG hyperstimulated group only when it was administered 24 h before hCG injection. Together, these results highlighted that 4-OHA, when administered at the appropriate time and dose, could reverse the negative effects of hyperstimulation from PMSG on early embryonic development. This may be due to its potent aromatase inhibiting properties that lead to the suppression of oestrogen production, thereby alleviating the supraphysiological level of oestradiol, which is typically present in PMSG-treated females. Interestingly, 4-OHA treatment on its own was able to positively influence embryo count when given at a high dose of 5000 microg/rat, and this may be associated with its weak androgenic properties. In conclusion, this study supports the hypothesis that excessive oestradiol is responsible for the negative effects of hyperstimulation with PMSG on early embryonic development.

The in vitro effects of low molecular weight serum fractions on embryonic Wistar rat (Rattus norvegicus) development.

The in vitro embryo culture technique has been used in many research areas as well as for teratologic and toxicologic tests. Usually, homologous serum is used as a culture medium in these techniques. In this study, the effects of low molecular weight serum fractions on embryonic rat development were tested. Using 30 and 50 kDa Macrosep centrifugal concentrators, the homologous serum was centrifuged for 8 h to separate the low molecular weight serum fractions. The embryos were cultured in the sera which included > 30 kDa, > 50 kDa and < 30 + > 50 kDa serum fractions. Whole rat serum (WRS) was also used for control. After a 48-h culture period, embryonic growth and development were assessed using a morphologic scoring system and the protein content of embryos and yolk sacs. The results showed that the embryonic growth and development during organogenesis significantly decreased in > 30 kDa and > 50 kDa serum fractions when compared to WRS. Addition of a < 30 kDa serum fraction to the > 50 kDa serum fraction improved the embryonic growth, but not to the level seen in embryos grown in WRS. While morphological scores for the embryos grown in WRS, > 30 kDa, > 50 kDa and < 30 + > 50 kDa serum fractions were 57.5 +/- 0.83, 44.53 +/- 1.06, 37.81 +/- 1.9 and 45.14 +/- 1.56, respectively, somite numbers were 26.4 +/- 0.28, 20.47 +/- 0.46, 19.15 +/- 0.58 and 21.78 +/- 0.5, yolk sac diameters were 3.35 +/- 0.06, 2.89 +/- 0.05, 2.61 +/- 0.03 and 2.71 +/- 0.04 mm, crown-rump lengths were 3.03 +/- 0.06, 2.72 +/- 0.04, 2.36 +/- 0.04 and 2.52 +/- 0.04 mm, embryo protein contents were 160.93 +/- 6.88, 119.07 +/- 5.15, 67.23 +/- 3.87 and 98.72 +/- 4.87 micrograms and yolk sac protein contents were 114.87 +/- 5.18, 86.33 +/- 1.92, 62.38 +/- 2.7 and 75.88 +/- 2.87 micrograms, respectively. These results suggest that low molecular weight serum fractions could be very important for normal embryonic development.

Cell Type- and Differentiation Stage-Dependent Expression of PML Domains in Rat, Detected by Monoclonal Antibody HIS55

Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In isolated rat liver nuclei, HeLa cells, and normal human and rat lymph nodes, staining of HIS55 colocalized with that of 5E10. 5E10 recognizes PML nuclear domains, multimolecular complexes of unknown function containing the product of PML gene and at least two other components. HIS55 foci were expressed widely in many tissues but the expression level varied in a cell type-specific manner, with the number of HIS55 nuclear foci ranging from 0 (as in neurons) to over 100 (as in megakaryocytes) and the size ranging from fine (as in cortical thymocytes) to very large (as in urethra epithelium). HIS55 ag expression level also varied among cells of the same lineage, as observed in embryonic development of rat and in the hemopoietic system of adult rat. The expression level of HIS55 loci roughly correlated with the overall rate of protein synthesis of cells, supporting a role of PML domains as transcription regulatory units. The expression of HIS55 foci, however, did not correlate with the growth index of cell populations. Our observations on normal tissues agreed with the hypothesis that PML domain expression is regulated by external, possibly site-dependent factors. We further supported this by demonstrating that PML domains in rat ventral prostate epithelia were upregulated upon castration.

Relationship of Migrating Luteinizing Hormone-Releasing Hormone Neurons to Unique Olfactory System Glycoconjugates in Embryonic Rats

Following their birth in olfactory placode, luteinizing hormone-releasing hormone (LHRH)-containing neurons migrate across the developing cribriform plate and form a dispersed population in the mammalian basal forebrain. The present study reveals the colocalization of unique glycoconjugate antigens (detected with monoclonal antibody CC2) on a subset of LHRH-immunoreactive (LHRHir) cell bodies and growth cones in the rostral forebrain during embryonic development in rats. In addition, LHRHir neurons were found along CC2-immunoreactive (CC2ir) fibers in the nasal cavity, across the cribriform plate, and in the rostral forebrain. At embryonic Day 16 (E16) approximately 20% of the LHRHir neuronal population in the forebrain had the CC2 epitope on surfaces of cell bodies. This percentage fell as the number of LHRHir neurons in the forebrain increased. Prior to the detection of LHRH-containing neurons, beginning on E14, CC2ir glycoconjugates were observed on vomeronasal cells and axons and also on a dorsomedial subset of olfactory neurons and axons. As early as E14 CC2ir fibers extended into the rostral forebrain. LHRHir neurons were seen in close apposition to CC2ir fibers in both the nasal cavity and rostral forebrain. These studies raise the possibility that CC2ir glycoconjugates provide a specific chemical guide for a subset of LHRH neurons along a part of their migratory pathways. The small percentage of LHRHir neurons which have CC2ir on their surfaces prenatally may constitute a selective homogenous functional subgroup within the population of LHRH neurons.

Cells Expressing mRNA for Neurotrophins and their Receptors During Embryonic Rat Development

In situ hybridization analysis of cells expressing messenger RNAs (mRNAs) for the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their high-affinity receptors (trk, trkB and trkC) in the rat embryo revealed a complex but specific expression pattern for each of these mRNAs. For all mRNAs a developmentally regulated expression was seen in many different tissues. BDNF and NT-3 mRNAs were expressed in the sensory epithelia of the cochlea and vestibule macula of the sacculus and utricle, and both trkB and trkC mRNA were expressed in the spiral and vestibule ganglia innervating these sensory structures. NGF and NT-3 mRNA were found in the iris, innervated by the sympathetic neurons of the superior cervical ganglion and sensory neurons from the trigeminal ganglion, which expressed both trk and trkC mRNAs. Both NGF and NT-3 mRNAs were also expressed in other target fields of the trigeminal ganglion, the epithelium of the whisker follicles (NT-3 mRNA) and in the epithelium of the nose, tongue and jaw. NT-3 mRNA was found in the cerebellar external granule layer and trkC mRNA in the Purkinje layer of the cerebellar primordia. These sites of synthesis are consistent with a target-derived neurotrophic interaction for NGF, BDNF and NT-3. However, in some cases mRNAs for both the neurotrophins and their high-affinity receptors were detected in the same tissue, including the dorsal root, geniculate, superior, jugular, petrose and nodose ganglia, as well as in the hippocampus, frontal cortical plate and pineal recess, implying a local mode of action. Combined, these data suggest a broad function for the neurotrophins and their receptors in supporting neural innervation during embryonic development. The results also identify several novel neuronal systems that are likely to depend on the neurotrophins in vivo.

Effects of oxygen concentration on embryonic development in rats: a light and electron microscopic study using whole-embryo culture techniques.

By using a whole-embryo culture technique (New 1978), the effects of oxygen concentration (5%, 20% and 95% oxygen) on embryonic development in the rat were investigated by light and electron microscopy. The best embryonic development occurred when the 9.5-day-old embryos were cultured for 24 h with 5% oxygen, and the 10.5-day-old embryos with 20% oxygen (optimum oxygen concentration). When the 9.5- and 10.5-day-old embryos were cultured for 24 h with too little or too much oxygen, retardation of the embryonic growth and abnormal development was observed. Using light microscopy, numerous degenerating cells, exhibiting granular deposits in the cytoplasm, were seen, but the distribution of the degenerating cells was quite different between the two groups. With electron microscopy, the most striking feature of the degenerating cells in the embryos cultured with too little oxygen, was the extreme swelling of the mitochondria without any morphological alterations of the nucleus or the other cell organelles. On the other hand, the characteristic feature of the degenerating cells in the embryos exposed to too much oxygen, was the formation of phagolysosomes in the cytoplasm. Morphological alterations of the nucleus or mitochondria were not evident. In the present study, the possible teratogenic mechanism of too much or too little oxygen in the whole-embryo culture of the rat embryo is discussed.

Effect of centchroman on tubal transport and preimplantation embryonic development in rats.

A single oral administration of centchroman (1.25 mg/kg) to adult female rats within 24 h of mating induced slight acceleration in the rate of transport of embryos through the oviducts. The compound did not seem to produce any deleterious effect on preimplantation embryonic development since well organized and apparently normal embryos were collected from the genital tract up to Day 12 of pregnancy. The recovery rate of embryos from centchroman-treated rats was, however, significantly reduced after Day 4 of pregnancy. There was some stimulation in the rate of cleavage of embryos and morula to blastocyst transformation, but retardation in the shedding of the zona pellucida. The rate of blastocyst formation was not altered when 6-8-cell embryos collected from the oviducts of control rats were transferred to the uteri of control or centchroman-treated females. A delay in zona shedding was observed in the centchroman-treated recipients.

Molecular forms and solubility of acetylcholinesterase during the embryonic development of rat and human brain

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) form homologous sets of multiple molecular forms. The central nervous system of mammals contains mostly tetramers (G 4) and monomers (G 1). Their proportions have been shown to vary during maturation in rat brain. In order to examine whether a similar evolution occurs in the human, we performed parallel studies of the activity, solubility and molecular forms of acetylcholinesterase in rat and human brains at various stages. We find both similarities and differences: in rat brain, the enzyme increases mostly postnatally but in human brain acetylcholinesterase reaches a maximum at birth. There is an increase in the proportion of G 4 and a decrease in the solubility of this form in the absence of detergent in human as well as in rat brain. These changes occur around birth in rat, but during early pregnancy, before 11 weeks in human brain. In both species, the solubility of the enzyme in detergent-free buffers decreases progressively from more than 50% before birth to about 10–20% in the adult. In addition we analyzed butyrylcholinesterase as well as the levels of the neuron-specific enolase and of the glial S-100 protein. In human, γγ-enolase rises to its adult level after birth, but before the S-100 protein.

Inherited disorders in the regulation of serum calcium in rats raised from parathyroidectomised mothers.

IN a series of experiments to study the influence of maternal endocrine dysfunction on the embryonic development in rats, we have found that the first generation (F1) rats born to parathyroidectomised (Px) mothers possess a slightly, but significantly, lower serum calcium level than in normal control rats1. Furthermore, the serum calcium level in the rats of the third or fourth generations (F3 or F4) developed by brother–sister mating was still at a subnormal level. To characterise calcium metabolism in these animals, the response of serum calcium to removal of the parathyroid gland was examined in the F1, F3 and F4 rats. We report here that F3 and F4 rats raised from mothers parathyroidectomised on the 5th day of pregnancy showed a less marked decline in serum calcium level following parathyroidectomy as shown in the first generation, suggesting that the functional alteration resulted from the intrauterine hypocalcaemia persists in subsequent generations.

Effects of vitamin deficiencies on fertility, course of pregnancy, and embryonic development in rats

Experiments are reported on female rats fed a purified diet, adequate in protein, calories, and minerals, supplemented with water- and fat-soluble vitamins except for the individual vitamin being investigated. One of the four B vitamins, thiamine, riboflavin, pyridoxine, or pantothenic acid, was withheld from the animals for 35, 28, or 13 days before mating, and again supplied from the day of mating, or from the thirteenth day of gestation, or after birth of the young. In proportion to the duration of the deficiency there occurred decreased fertility, resorption or abortion, congenital malformations and serious abnormalities, and impaired growth of young.The 2,600 females were weighed daily, from the thirteenth day before mating until delivery of young, or the sacrifice of selected animals. Lowered fertility is indicated by a reduced rate of weight gain. Fluctuations in the individual weight curves appear to mark resorption or abortion. Congenital abnormalities were most frequent in fetuses obtained during the third week or in newborn of animals deficient in riboflavin during pregnancy. Those young carried to term in spite of maternal deficiencies are below normal birth weight, frequently show serious injuries, have a high postnatal mortality, and can rarely be weaned.Adequate supply of thiamine, riboflavin, pyridoxine, and pantothenic acid is necessary in rats to provide fertility, normal development of embryos, and the rearing of young through the weaning period.