Live Pups Research Articles

Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects.

Peri-ovulatory putrescine supplementation reduces embryo resorption in older mice.

Does peri-ovulatory putrescine supplementation of older mice improve oocyte quality and reduce the incidence of embryo resorption? Peri-ovulatory putrescine supplementation in older mice improved oocyte quality, as indicated by increased blastocyst cell numbers and reduced the incidence of embryo resorption. Rodents exhibit a transient rise of ornithine decarboxylase (ODC) and putrescine in the ovaries during ovulation. Older mice exhibit reduced ovarian ODC activity during ovulation. Supplementation of in vitro maturation medium with putrescine reduces oocyte aneuploidy rates of older mice. The rationale was to correct ovarian putrescine deficiency in older mice by peri-ovulatory putrescine supplementation in drinking water and to observe the reproductive consequences of this intervention. This project was conducted between 2010 and 2014. Older mice (9-11 months of age) were given regular drinking water (control) or drinking water with 1% putrescine dihydrochloride (62 mM) for 2-4 days before mating. Plugged mice were then withdrawn from putrescine supplementation. Blastocysts were retrieved on 3.5 days post coitum (dpc) for the determination of cell numbers. For resorption analyses, mice were killed on 9.5 dp or 12.5 dpc, and implantation sites were dissected to determine the embryo status. For birth studies, mice were examined every morning between 16.5 and 23.5 dpc. Births were recorded as live or stillbirth. We demonstrated that deficiency of ovarian putrescine in older mice can be restored by peri-ovulatory putrescine supplementation in drinking water. Putrescine supplementation in older mice increased blastocyst cell numbers (from 40 to 54; P < 0.0001, t-test), reduced embryo resorption rates (from 41.1 to 15.4% in old C57BL/6 mice, P < 0.0001, Fisher's exact test; from 14.2 to 6.4% in old CF1 mice, P = 0.004, Fisher's exact test), and doubled the number of live born pups. Furthermore, exogenous putrescine exhibited rapid absorption and excretion, and showed no toxicity to mothers or fetuses. The mechanism of putrescine action in oocytes and/or ovaries remains unclear. Peri-ovulatory putrescine deficiency in older mice appears to adversely impact on oocyte maturation resulting in poor quality embryos (as assessed by blastocyst cell numbers) and early embryo death. This study demonstrates a natural and simple remedy to improve oocyte quality in older women. This study was supported by the NSERC, the March of Dimes Foundation, and the National Natural Science Foundation of China. The authors declare no competing interest.

Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice.

Superovulation is a reproductive technique generally used to produce genetically engineered mice. Superovulation in mice involves the administration of equine chorionic gonadotropin (eCG) to promote follicle growth and then that of human chorionic gonadotropin (hCG) to induce ovulation. Previously, some published studies reported that inhibin antiserum (IAS) increased the number of ovulated oocytes in ddY and wild-derived strains of mice. However, the effect of IAS on the C57BL/6 strain, which is the most widely used inbred strain for the production of genetically engineered mice, has not been investigated. In addition, the combined effect of IAS and eCG (IASe) on the number of ovulated oocytes in superovulation treatment has not been examined. In this study, we examined the effect of IAS and eCG on the number of ovulated oocytes in immature female mice of the C57BL/6 strain in superovulation treatment. Furthermore, we evaluated the quality of obtained oocytes produced by superovulation using IASe by in vitro fertilization (IVF) with sperm from C57BL/6 or genetically engineered mice. The developmental ability of fresh or cryopreserved embryos was examined by embryo transfer. The administration of IAS or eCG had a similar effect on the number of ovulated oocytes in C57BL/6 female mice. The number of ovulated oocytes increased to about 3-fold by the administration of IASe than by the administration of IAS or eCG alone. Oocytes derived from superovulation using IASe normally developed into 2-cell embryos by IVF using sperm from C57BL/6 mice. Fresh or cryopreserved 2-cell embryos produced by IVF between oocytes of C57BL/6 mice and sperm from genetically engineered mice normally developed into live pups following embryo transfer. In summary, a novel technique of superovulation using IASe is extremely useful for producing a great number of oocytes and offspring from genetically engineered mice.

A Mouse Model of Hypospadias Induced by Estradiol Benzoate.

We wished to establish a mouse model of hypospadias using injections of estradiol benzoate for investigating the molecular mechanisms of hypospadias. Fifty timed pregnant mice were randomly divided into five study groups: A, B, C, D, and E. These groups were injected subcutaneously with estradiol benzoate mixed with sesame oil at, respectively, the doses of 0, 0.1, 0.5, 2.5, or 12.5mgkg(-1)days(-1) from gestation day (GD) 12 to GD 16. The pups' mortality was recorded on the day of delivery. Urethras and positions of testes were examined on postnatal day 28. The numbers of live pups were significantly lower in the study groups D and E compared to study group A (p<0.01). Hypospadias was seen in groups C (3.3%; 1/30), D (18.2%; 4/22), and E (21.4%; 3/14), while cryptorchidism was observed in groups C (10%; 3/30), D (31.8%; 7/22), and E (57.1%; 8/14) on postnatal day 28. The experimental model of hypospadias induced by estradiol benzoate in the group D (2.5mgkg(-1)days(-1)) was more reliable considering high mortality of the study group E. The dose of estradiol benzoate used in the group D is suitable for establishing mouse model of hypospadias.

Effects of prolonged exposure of mouse embryos to elevated temperatures on embryonic developmental competence

To investigate effects of heat stress on developmental competence, in-vitro fertilized zygotes were incubated at different temperatures until 96 h post human chorionic gonadotrophin (HCG). Under severe and moderate conditions (41°C and 40°C), most embryos did not overcome the 2-cell block. In long-term mild heat stress (39°C until 96 h post HCG), cleavage and blastocyst formation were comparable to non-heat-stress control, but the number of live pups per transferred embryo and mean litter size were significantly affected (P < 0.05) in the mild-heat-stress group (19.4%, and 5.1 ± 0.4, respectively), compared with control (41.7% and 8.3 ± 0.3, respectively). To elucidate the different competence, gene expression was examined and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells were counted. Aberrant expression of genes for embryonic viability and trophoblast differentiation in the mild-heat-stressed blastocysts was found. Moreover, the expanded blastocysts in the heat-stressed group and the control had a ICM:TE ratio of 1:2.47 and 1:2.96 with average total cell numbers of 59.21 ± 2.38 and 72.79 ± 2.40, respectively (P < 0.05), indicating lower cell numbers in TE. These findings underscore that prevention of heat stress in early embryos is important for maintaining embryo viability embryos during pregnancy.

Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9.

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.

Heterozygote advantage at MHC DRB may influence response to infectious disease epizootics.

The effect of MHC polymorphism on individual fitness variation in the wild remains equivocal; however, much evidence suggests that heterozygote advantage is a major determinant. To understand the contribution of MHC polymorphism to individual disease resistance or susceptibility in natural populations, we investigated two MHC class II B loci, DQB and DRB, in the New Zealand sea lion (NZSL, Phocarctos hookeri). The NZSL is a threatened species which is unusually susceptible to death by bacterial infection at an early age; it has suffered three bacterial induced epizootics resulting in high mortality levels of young pups since 1997. The MHC DQB and DRB haplotypes of dead NZSL pups with known cause of death (bacteria, enteritis or trauma) were sequenced and reconstructed, compared to pups that survived beyond 2months of age, and distinct MHC DRB allele frequency and genotype differences were identified. Two findings were striking: (i) one DRB allele was present only in dead pups, and (ii) one heterozygous DRB genotype, common in live pups, was absent from dead pups. These results are consistent with some functional relationship with these variants and suggest heterozygote advantage is operating at DRB. We found no association between heterozygosity and fitness at 17 microsatellite loci, indicating that general heterozygosity is not responsible for the effect on fitness detected here. This result may be a consequence of recurrent selection by multiple pathogen assault over recent years and highlights the importance of heterozygote advantage at MHC as a potential mechanism for fitness differences in wild populations.

Maternal Magnesium Supplementation Reduces Intrauterine Growth Restriction and Suppresses Inflammation in a Rat Model

Objective Intrauterine growth restriction (IUGR) is associated with increased inflammatory responses. We sought to investigate whether magnesium (Mg) attenuates inflammation and IUGR in a rat model. Study design Pregnant Wistar rats (12 weeks, gestational day 18) were randomly assigned to 1 of 4 groups: normal diet with bilateral uterine artery ligation (BL) (n = 6) or sham surgery (SH) (n = 5); and Mg chloride (MgCl 2 ) 1% (wt/vol) in the drinking water throughout gestation + BL (MgBL) (n = 6) or SH (MgSH) (n = 5). Dams were euthanized 24 hours postsurgery (gestational day 19). Maternal plasma, fetal plasma (pooled), individual amniotic fluid (AF) samples, and placentas (PL) were collected and assessed from live fetal pups only (BL, n = 36; SH, n = 20; MgBL, n = 20; MgSH, n = 20). All samples were analyzed for cytokines/chemokines (interleukin [IL]-6, IL-1β, chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-C motif] ligand 2 [CCL2], and tumor necrosis factor [TNF-α] sensitivity Results The incidence of IUGR (pup weight P P P P P 2 supplementation significantly decreased IL-1β, TNF-α, and CCL2 levels in AF and IL-1β in PL tissues of MgBL vs BL rats ( P Conclusion Maternal oral MgCl 2 supplementation reduced BL-induced IUGR by 64% and suppressed cytokine/chemokine levels in the AF and PL.

Tauroursodeoxycholic acid improves the implantation and live-birth rates of mouse embryos

We previously demonstrated that tauroursodeoxycholic acid (TUDCA) improved the developmental competence of mouse embryos by attenuating endoplasmic reticulum (ER) stress-induced apoptosis during preimplantation development. Here, we present a follow-up study examining whether TUDCA enhances the implantation and live-birth rate of mouse embryos. Mouse 2-cell embryos were collected by oviduct flushing and cultured in the presence or absence of 50μM TUDCA. After culture (52h), blastocysts were transferred to 2.5-day pseudopregnant foster mothers. It was found that the rates of pregnancy and implantation as well as the number of live pups per surrogate mouse were significantly higher in the TUDCA-treated group compared to the control group, but there was no significant difference in the mean weights of the pups or placentae. Thus, we report for the first time that TUDCA supplementation of the embryo culture medium increased the implantation and livebirth rates of transferred mouse embryos.

Administration of the nonsteroidal anti-inflammatory drug tolfenamic acid at embryo transfer improves maintenance of pregnancy and embryo survival in recipient mice.

To evaluate the effect of the nonsteroidal anti-inflammatory drugs tolfenamic acid and flunixin meglumine in pregnancy rate and embryo survival of recipient mice subjected to embryo transfer. A total of 142 recipient females were transferred with 2,931 embryos and treated with a single injection of tolfenamic acid (1 mg/kg; n = 54 females with 1,129 embryos), flunixin meglumine (2.5 mg/kg; n = 46 females with 942 embryos), or bi-distilled water (10 mL/kg) as control group (n = 42 females with 860 embryos). Pregnancy was checked 2 weeks after embryo transfer, delivery was registered on the due date, and litter size was recorded on Day 7 after birth. Pregnancy rate of tolfenamic acid treated females was significantly higher than flunixin group (P < 0.05) and showed a tendency to be higher when compared to the control group (P = 0.06). The number of pups born from transferred embryos in pregnant females was significantly higher for both treatment groups compared to controls (P < 0.05). Number of pups from total transferred embryos was higher for both treatment groups (P < 0.05) when compared to controls. The use of tolfenamic acid at the time of embryo transfer improves both pregnancy rate and number of live pups in recipient mice, with optimal effects observed with flunixin meglumine. We suggest that the use of tolfenamic acid has beneficial effects on the maintenance of pregnancy and embryo survival in recipient mice, which should be taken into account for further studies in other mammalian females.

Prevalence of Presenting Conditions in Grey Seal Pups (Halichoerus grypus) Admitted for Rehabilitation.

A retrospective survey was performed on the presenting conditions of 205 live grey seal pups (Halichoerus grypus) admitted to the Cornish Seal Sanctuary in Gweek, United Kingdom between May 2005 and March 2011. The purpose of the survey was to examine the prevalence of various presenting signs at the sanctuary. The presenting signs were classified into nine non-mutually exclusive categories: ocular disorders, nasal disorders, oral disorders, respiratory disorders, orthopaedic disorders, puncture wounds, abrasions, netting injuries, and onychia. The sex ratio of seal pups in this study was 1.35 males per female. Of the 205 examined for rehabilitation, 22 (10.73%) did not survive to release. 68.78% of grey seal pups presented with puncture wounds, 47.80% with respiratory disorders, 46.34% with ocular disorders, 42.63% malnourished, 36.59% with abrasions, 25.37% with oral disorders, 23.90% with nasal disorders, 11.71% with orthopaedic disorders, 9.27% with onychia, and 3.41% presented with netting injuries. 52% were normothermic, 42% were hyperthermic, and 5% were hypothermic. Associations between gender, outcome of rehabilitation, hospitalisation time and presenting disorders were examined. In addition, admissions rates were found to display seasonality. The results of this study will aid in future preparation of grey seal rehabilitation facilities.

Evidence of land-sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species.

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C.jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole-genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype-host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.

Latitudinal variation in local productivity influences body condition of South American sea lion pups

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 23:39-47 (2014) - DOI: https://doi.org/10.3354/ab00604 Latitudinal variation in local productivity influences body condition of South American sea lion pups Maritza Sepúlveda1,2,*, Danai Olea1, Pablo Carrasco3,4, Macarena Santos-Carvallo1,2, Jorge Castillo5, Renato A. Quiñones3,4 1Centro de Investigación y Gestión de los Recursos Naturales (CIGREN), Instituto de Biología, Facultad de Ciencias, Universidad de Valparaíso, Gran Bretaña 1111, Playa Ancha, Valparaíso 2360102, Chile 2Centro de Investigación Eutropia, Ahumada 131 Oficina 912, Santiago 8320238, Chile 3Programa de Investigación Marina de Excelencia (PIMEX), Facultad de Ciencias Naturales y Oceanográficas, Casilla 160-C, Universidad de Concepción, Concepción 4070205, Chile 4Interdisciplinary Center for Aquaculture Research (INCAR-FONDAP), Universidad de Concepción, O’Higgins 1695, Concepción 4070007, Chile 5Instituto de Fomento Pesquero, Casilla 8v, Valparaíso, Almte. Manuel Blanco Encalada 839, Valparaíso 2361827, Chile *Corresponding author: maritza.sepulveda@uv.cl ABSTRACT: In otariids, the condition and growth of offspring are linked to prey availability because females show a strong dependence on local food availability. Thus, it is expected that body condition of sea lion pups will vary spatially and/or temporally as a response to variations in the abundance of prey species on which females feed. The objective of this study was to analyze the geographic and temporal variation in the body condition of South American sea lion Otaria flavescens pups in Chile, and to relate it to spatio-temporal variations in prey availability. We captured 340 live pups in 2 distant colonies, Punta Patache/Punta Negra and Cobquecura, along the Chilean coast during consecutive breeding seasons. A morphometric index of pup body condition was estimated by comparing pups in all years using least-squares linear regression of the log10‑transformed measurements of standard length vs. body mass. We analyzed the relationship between this index and estimates of fish biomass (as a proxy of prey availability) at each locality. We found that body condition was significantly different between years and between colonies, suggesting that animals of the central-south area were in better condition than those in the north. A positive relationship between body condition and fish biomass was found, suggesting that differences in body condition may be explained by spatial and temporal differences in prey availability. KEY WORDS: Body condition index · Body mass · Environmental effects · Morphometrics · Otaria flavescens · Southern sea lion Full text in pdf format PreviousNextCite this article as: Sepúlveda M, Olea D, Carrasco P, Santos-Carvallo M, Castillo J, Quiñones RA (2014) Latitudinal variation in local productivity influences body condition of South American sea lion pups. Aquat Biol 23:39-47. https://doi.org/10.3354/ab00604 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 23, No. 1. Online publication date: December 02, 2014 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2014 Inter-Research.

EVALUATION OF REPRODUCTIVE PERFORMENCE OF DOVES AFTER TREATMENT WITH INSEMINATION DOSE IMPLEMENTER HEPARIN

The aim of the study was to evaluate the effect of implementer heparin on the interaction follicles - sperm, conceptual ratio and the number of pups born in inseminated doves inseminated The experimental group was inseminated with 0.5 ml insemination dose inseminated 15 min. incubation with heparin (25 000 IU / l ml) of concentrations of 0.06 ml / 0.5 ml / ID (0.06 ml = 10 mg). The control group was inseminated with 0.5 ml insemination dose without implementer. The addition of heparin implementer has led to an increase in the reproductive performance in vitro and in vivo. Number of zygotes with two pronucleus represented in the control group 74.5% of the total number of follicles stranded in the group. In the experimental group, the number of zygotes with two pronucleus accounted for 81.08% of the total number of follicles stranded in the group. In vivo, we found a conceptual ratio 89.28% with an average number of live pups per litter 10.64 ± 3.41 pc in experimental doves. In the control group the conceptual ratio was 72.00% with an average number of pups per litter 8.55 ± 3.22 pc. The conceptual relation between the control K and experimental GAG group of doves has show a statistically significant difference (χ24, 15 +). In the number of live born pups between control K and experimental GAG group a statistically significant difference (2.02 + T41) was observed too.

Epidemiology of hookworm (Uncinaria sanguinis) infection in free-ranging Australian sea lion (Neophoca cinerea) pups.

Understanding the fundamental factors influencing the epidemiology of wildlife disease is essential to determining the impact of disease on individual health and population dynamics. The host-pathogen-environment relationship of the endangered Australian sea lion (Neophoca cinerea) and the parasitic hookworm, Uncinaria sanguinis, was investigated in neonatal pups during summer and winter breeding seasons at two biogeographically disparate colonies in South Australia. The endemic occurrence of hookworm infection in Australian sea lion pups at these sites was 100% and post-parturient transmammary transmission is likely the predominant route of hookworm infection for pups. The prepatent period for U. sanguinis in Australian sea lion pups was determined to be 11-14 days and the duration of infection approximately 2-3 months. The mean hookworm infection intensity in pups found dead was 2138 ± 552 (n = 86), but a significant relationship between infection intensity and faecal egg count was not identified; infection intensity in live pups could not be estimated from faecal samples. Fluctuations in infection intensity corresponded to oscillations in the magnitude of colony pup mortality, that is, higher infection intensity was significantly associated with higher colony pup mortality and reduced pup body condition. The dynamic interaction between colony, season, and host behaviour is hypothesised to modulate hookworm infection intensity in this species. This study provides a new perspective to understanding the dynamics of otariid hookworm infection and provides evidence that U. sanguinis is a significant agent of disease in Australian sea lion pups and could play a role in population regulation in this species.

Efficient generation of sFat-1 transgenic rabbits rich in n-3 polyunsaturated fatty acids by intracytoplasmic sperm injection.

N-3 polyunsaturated fatty acids (n-3 PUFAs) have their first double bond at the third carbon from the methyl end of the fatty-acid chain and had been proven to be beneficial to human health. However, mammals cannot produce n-3 PUFAs by themselves because they lack the n-3 fatty-acid desaturase (Fat-1) gene. Thus, the possibility of producing sFat-1 transgenic rabbits was explored in this study. The transgenic cassette of pPGK1-sFat-1-CMV-EGFP was constructed and transgenic rabbit embryos were produced by intracytoplasmic sperm injection (ICSI). When 123 EGFP-positive embryos at the 2-8-cell stage were transplanted into the oviduct of four oestrous-synchronised recipients, two of them became pregnant and gave birth to seven pups. However, transfer of embryos into the uterus of oestrous-synchronised recipients and oviduct or uterus of oocyte donor rabbits did not result in pregnancy. The integration of the sFat-1 gene was confirmed in six of the seven live pups by PCR and Southern blot. The expression of the sFat-1 gene in the six transgenic pups was also detected by reverse transcription polymerase chain reaction (RT-PCR). Gas chromatography-mass spectrometry analysis revealed that transgenic rabbits exhibited an ~15-fold decrease in the ratio of n-6:n-3 PUFAs in muscle compared with wild-type rabbits and non-transgenic rabbits. These results demonstrate that sFat-1 transgenic rabbits can be produced by ICSI and display a low ratio of n-6:n-3 PUFAs.

De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes

The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials.

Dissecting aortic aneurysm induced by N-(2-aminoethyl) ethanolamine in rat: Role of defective collagen during development.

Dissecting aortic aneurysm (DAA) is a tear in the wall of the aorta that causes blood to flow, or "dissect," between the medial layers of the media. Pregnant rats (dams) were treated with the industrial chemical n-(2-aminoethyl) ethanolamine (AEEA) by intraperitoneal injection or gavage. The histology and pathology of aorta in the thorax from newborn pups were examined. Aortas of fetuses of gestational day 20 from dams exposed to AEEA were harvested for immunohistochemical staining and native Western blot to study the changes of collagen type 1 and type 3 in aorta. Dissecting aortic aneurysm of newborn rats was induced by treating with AEEA through intraperitoneal injection or gavage. The incidence of DAA reached 100% in live pups at the high dose by means of gavage of AEEA, but without lethality compared with intraperitoneal injection. A grading system for the dose-response of DAA lesions associated with AEEA by gavage was established. Gestational day 20 fetuses from treated dams showed a decreased content and altered distribution of medial and adventitial collagen type 1 and 3 in aorta by immunohistochemistry; this decrease was confirmed by native Western blot. This in vivo model of spontaneous aortic dissection bears striking similarities histologically to human aortic dissection. As such, the model conceivably could contribute to elucidating the mechanisms of DAA formation and to exploring diagnostic and therapeutic strategies. The pathogenesis of AEEA-induced DAA may be related to defects in the normal developmental progression of collagen types 1 and 3 in the vascular wall.

De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes.

The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials.

Nicotine, an α7 nAChR agonist, reduces lipopolysaccharide-induced inflammatory responses and protects fetuses in pregnant rats

The objective of the study was to examine the effects of nicotine, an α7 nicotinic acetylcholine receptor agonist, on lipopolysaccharide (LPS)-induced inflammatory responses in rats during pregnancy. Pregnant Sprague Dawley rats were randomly divided into groups (n = 6 rats/group): group 1 rats each received a single intraperitoneal injection of LPS (25 μg/kg) on gestation day 16; group 2 rats were first pretreated with nicotine (1 mg/kg per day, subcutaneously) on gestation days 14 and 15 and then were treated with single injections of LPS on gestational day 16; group 3 rats were treated with the vehicle (saline) used for groups 2 and 3 (controls). Maternal blood was collected at 6 hours and 24 hours after LPS and vehicle treatments and assayed for tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and interleukin-10 (IL-10). In addition, the number of live pups and pup weights were obtained at the time of delivery. LPS treatment significantly (P < .001) elevates maternal blood levels of TNF-α and IL-6 but not IL-10 (P > .05). Nicotine treatment significantly reduces LPS-induced TNF-α and IL-6 concentrations (P < .001) but does not change (P > .05) IL-10 levels. The number of live pups in the LPS group are significantly lower (P < .001) than the vehicle treated controls, and nicotine treatment significantly (P < .011) reverses this change. Similarly, fetal weights are lower following LPS (P < .016) and higher (P < .024) in the group treated with nicotine plus LPS. Nicotine reduces the LPS-induced inflammatory responses and rescues the fetus in rats during pregnancy. Thus, nicotine exerts dramatic antiinflammatory effects. These observations have important implications for control of inflammatory responses during pregnancy.