Pseudopregnant Females Research Articles

PGFM (13,14-dihydro-15-keto-PGF 2α) in pregnant and pseudo-pregnant Iberian lynx: A new noninvasive pregnancy marker for felid species

In mammals, uterine and placental prostaglandin F 2α is involved in the regulation of reproduction-related processes such as embryonic development, initiation of parturition, and resumption of ovarian activity. Prostaglandin F 2α (PGF 2α) is rapidly metabolized to its plasma metabolite PGFM (13,14-dihydro-15-keto-PGF 2α), which has also been detected in urine. Therefore, the current study aimed to develop and validate an efficient, quick, and inexpensive enzyme immunoassay (EIA) for PGFM estimation in urine of the Iberian lynx ( Lynx pardinus) for pregnancy monitoring and for differentiation between pregnancy and pseudo-pregnancy. Urine samples collected from captive Iberian lynx (11 pregnant and 4 pseudo-pregnant cycles) were subjected directly to a PGFM EIA. The assay was validated for parallelism, precision, and stability of urinary PGFM. In addition, high-performance liquid chromatography (HPLC) immunograms and liquid chromatography–mass spectrometry (LCMS) were performed to identify PGFM within urine samples. Urinary PGFM levels before mating and after parturition were about 1.5 ng/mL. After Day 20 postmating, both pregnant and pseudo-pregnant females showed slight increase of hormone levels; in pseudo-pregnant females, this elevation did not exceed 7 ng/mL. A significant increase in pregnant females was observed after Day 45 postmating; urinary PGFM increased from 10 ng/mL at Day 45 toward a peak of 46.0 ± 19.3 ng/mL around parturition. First results show that PGFM is detectable in feces as well and follows similar courses as shown for urine. In conclusion, the presented and validated PGFM assay is an easy and reliable method for noninvasive pregnancy diagnosis in the Iberian lynx (and probably other felids) if applied approximately 20 d prior parturition in pure urine or fecal extracts. High PGFM levels in urine or fecal samples may allow a pregnancy diagnosis without knowledge of mating time, making the PGFM test applicable to free-ranging animals.

Establishment and Characterization of A New Leukemia Mouse Model Induced by MLL/AF4 Fusion Protein.

Abstract 3573Poster Board III-510 BackgroundMLL/AF4 fusion is the most common abnormality in infant leukemia and is associated with a poor prognosis. Although further study and treatment should be groped for acute lymphoblastic leukemia (ALL) with MLL/AF4, the lack of a proper animal model has impeded understanding of the molecular mechanism of leukemia associated with the MLL/AF4 fusion gene. Previous mouse models suggest that the tumorigenesis capacity of MLL/AF4 alone is insufficient for development mixed-lineage leukemia. MLL/AF4 is capable of inducing lymphoid malignancy with long latency, which is very different from aggressive pro-B ALL in humans. Recently, we have reported that enhancement of S100A6 (calcyclin) expression played an important role in MLL/AF4-leukemogenesis using murine 32Dc cell line. In this study, we tried to make improved MLL/AF4 transgenic (Tg) mice which reflect human leukemogenicity and analyzed molecular mechanism of MLL/AF4 fusion gene. MethodsTo get high and sustained expression of MLL/AF4 in hematological lineage, we used MSCV promoter to express MLL/AF4 fusion gene. After MLL/AF4 expression plasmids were injected into fertilized eggs of mice (C57BL/6), the manipulated embryos were transferred into the oviducts of pseudopregnant females to make MLL/AF4 Tg mice. Finally we characterized MLL/AF4 Tg mice and analyzed molecular mechanism of MLL/AF4-leukemogenesis. ResultsOur new established MLL/AF4 Tg mice had lymphoma earlier than previous MLL/AF4 knock-in mice (median 12M vs 17M). Infiltration of lymphoid cells was located in liver, lung, kidney, and spleen and they consist of pro B-cell (CD45R+CD43+CD19-) lineage. Leukemic change developed two months (median 14M) after appearance of lymphoma. More than 30% of abnormal lymphocytes and severe anemia (RBC: 276×104 ml, Hb: 4.9 g/ml) were found in peripheral blood in MLL/AF4 Tg mice. Western blot analysis showed that up-regulation of HoxA9 and S100A6 was detected in MLL/AF4 Tg mouse compared to wild type C57BL/6. ConclusionWe succeeded in establishment of improved more aggressive MLL/AF4 Tg mice which reflect MLL/AF4 leukemogenicity of humans. Appearance of lymphoma in our MLL/AF4 Tg mice needed short er latency than those of the previous knock-in mice. Enhancement of HoxA9 and S100A6 expression was involved in MLL/AF4-associated leukemogenesis. We believe that this new MLL/AF4 Tg mouse is useful for study of the molecular mechanisms of MLL/AF4 leukemogenesis and development of new therapy for mixed-lineage leukemia. Disclosures:No relevant conflicts of interest to declare.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.

Transgenic animals in male reproduction research.

Although transgenic mouse technology has already been widely used for the study of gene function and regulation in many areas of biomedicine, it has been applied only sporadically to the investigation of testicular function. Nevertheless, the contribution of this experimental approach to the understanding of male reproduction is considerable, not least because of the frequency of infertility in transgenic mice. Transgenic mice can be produced by microinjection of DNA constructs in the male pronucleus of fertilized eggs that are then retransferred into the oviducts of pseudopregnant females and allowed to develop to term. A proportion of the offspring have the foreign DNA sequences permamently integrated into the genome and thus become transgenic. In this way it is possible to obtain either the over-expression of genes, which can be targeted to the testis using testis-specific promoters, or to effect interruption of the functional integrity of genes by insertional mutagenesis. The regulation of gene expression in vivo can be studied by producing transgenic mice where the transgene is composed of the regulatory sequences of a gene of interest driving the expression of a reporter gene. Specific genes can be "knocked out" by homologous recombination. This article reviews the contribution of the transgenic approach to the following areas of male reproduction: the identification of factors involved in sex determination and development of the reproductive tract; the study of the function and expression of genes important for spermatogenesis and male reproduction; the identification of genes involved in spermatogenesis and of genomic sequences directing the expression of a transgene in the testis; the study of the function of specific reproductive tissues or cells in vivo; oncogenesis in reproductive tissues; the creation of cell lines suitable for in vitro studies; gene therapy.

Effect of Stem Cell Activation, Culture Media of Manipulated Embryos, and Site of Embryo Transfer in the Production of F0 Embryonic Stem Cell Mice1

Recently, F0 embryonic stem (ES) cell mice have been produced by injection of ES cells into eight-cell embryos using either laser- or piezo-assisted injection systems. To simplify the injection procedure, we have optimized the conventional blastocyst injection method, free of laser- or piezo-assisted micromanipulation systems, to produce F0 ES cell pups. To increase the efficiency of producing mice from ES cell injection into eight-cell and blastocyst stage embryos, we have tested: 1) the effect of activating ES cell before injection, 2) the effect of in vitro culture in medium optimized for the survival of both ES cells and embryos, and 3) the effect of transferring the micromanipulated embryos into the oviduct versus into the uterus of CD1 foster mice. Two B6D2 hybrid ES cell lines were used for injection in a multifactorial analysis to evaluate the efficiency of producing live chimeric and F0 ES cell mice. Our results demonstrate that the activation of ES cells and the appropriate culture conditions are crucial parameters influencing the generation of F0 ES cell offspring. Transfer of blastocysts injected with ES cells into the oviduct of 0.5-day postcoitum pseudopregnant females increased the number of live animals with higher chimera proportion. Under these conditions, injections into eight-cell embryos produce a high number of F0 ES mice, and the conventional blastocyst injection method produces a lower number of F0 ES cell pups; however, the efficiency of production of chimeric mice with germline transmission was high. We have developed an economical and efficient technique for producing fully ES cell-derived F0 mice with full germline transmission that can be applied in many laboratories without the use of piezo or laser instruments.

Polo-like Kinase 2 (PLK2) Phosphorylates α-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.

Modulating the Expression of Imprinted Genes in Mouse Parthenogenetic Embryos

ABSTRACTThe effect of transforming growth factor α (TGFα) on the expression of imprinted genes Igf2, H19 and Pegl/Mest in diploid parthenogenetic mouse embryos (PE) from hybrids (CBA x C57BL/6)F1 in posimplantational period of embryonal development has been studied. PE were treated with TGFα in vitro at morula stage and after development to blastocyst stage were transplanted in the uterus of pseudopregnant females. At day 10 from the begining of pregnancy PE were explanted for culturing in vitro for 48 hours. The expression of imprinted genes Igf2, H19 and Peg1/Mest was studied by the methods RT-PCR u whole mount in situ RNA-RNA hybridization by labelled with digoxygenin antisens RNA probes. It has been demonstrated, that before beginning of culturing in 60% from treated PE at day 10 (stage of 21–25 somites) expression of Igf2 has been established only in forebrain, expression of Peg1/Mest has not been established and expression of H19 was significantly diminished. In the control non-treated with TGFα PE at the same stages (21–25 somites) expression of the genes Igf2 and Peg1/Mest has not been detected. After 48 hours culture in vitro in all PE treated with TGFα and developed to stage of 40–45 somites, expression of Igf2 has been established in the cerebrum, heart, forming maxillae, liver and somites, expression of Peg1/Mest was found in the cerebrum, heart and liver. In the control non-treated with TGFα PE at similar stages of development expression of Igf2 and Peg1/Mest was not detected. The data received shows, that exogenic TGFα is able to reactivate expression of two imprinted genes (Igf2 u Peg1/Mest) and to repress third imprinted gene (H19). In this way TGFα modulates the effects of genomic imprinting and improve significantly viability and development of PE. The possibility that gene coding for TGFα is imprinted is discussed‥

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Chromosome aberrations in mouse embryos and fetuses produced by assisted reproductive technology

The occurrence of structural chromosome aberrations in mouse one-cell embryos produced by intracytoplasmic sperm injection (ICSI) with mature spermatozoa was dependent on the type of sperm incubation medium and sperm incubation time. When cauda epididymal spermatozoa were used following incubation in bicarbonate-buffered TYH medium for 0 h (no incubation) and 0.5 h, the chromosome aberration rates (6.9% and 7.4%, respectively) in the resultant embryos were significantly higher than that (2.3%) in the IVF embryos. However, when the spermatozoa were incubated for 2–2.5 h and 6 h in the same medium, the chromosome aberration rates were reduced to the IVF embryo level (3.8% and 4.3%, respectively). When spermatozoa incubated in Hepes-buffered H-mCZB and phosphate-buffered PB1 media were used for ICSI, chromosome aberration rates in embryos were significantly high (8.6–28.1%) and increased in a time-dependent manner. On the other hand, when immature testicular spermatozoa were incubated in those three media for 0.5 h and 6 h, the incidences of resultant embryos with structural chromosome aberrations ranged between 7.4% and 11.7%, and there was no medium- and time-dependent change in these aberration rates. To evaluate transmissible risk of chromosome aberrations to offspring, two- or four-cell embryos derived from cauda epididymal spermatozoa were transferred into the oviducts of pseudopregnant females and chromosomes of live fetuses were examined on gestational day 16. One (2.0%) mosaic fetus was found when spermatozoa were incubated in TYH for 2–2.5 h, and there were four (6.7%) fetuses displaying a structurally abnormal karyotype when spermatozoa were incubated in H-mCZB for 2–2.5 h, indicating that structural chromosome aberrations generated in ICSI one-cell embryos are transmissible to offspring. The causal mechanism of structural chromosome aberrations in ICSI one-cell embryos is discussed in relation to the acrosomal plasma membrane cholesterol and the acrosome.

Non‐invasive Monitoring of Hormones: A Tool to Improve Reproduction in Captive Breeding of the Eurasian Lynx

The survival of many critical endangered mammal species is often depending on successful captive breeding programmes which include the future option of reintroduction to the wild. Breeding in captivity also demands the application of modern assisted reproductive techniques to ensure maximal biodiversity, but knowledge on reproductive physiology is often limited. Therefore, non-invasive monitoring of urinary and faecal hormones has become an important tool for reproductive management. To exemplify the importance of non-invasive hormone monitoring, we choose the Eurasian lynx as a model for the world's most endangered felid species, the Iberian lynx. We analysed faecal samples of pregnant and pseudo-pregnant female Eurasian lynxes during a 3-year study period. Compared to pre-mating levels faecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Oestrogen levels raised in both pregnant and pseudo-pregnant females with a tendency to be more elevated and prolonged in pregnant females. Surprisingly both E2 and P4 metabolites were highly correlated (r(2) =0.8131, p < 0.0001) showing a postpartum increase both in pregnant and pseudo-pregnant females. The results from the Eurasian lynx revealed that the measurement of faecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species, but similar to those from faecal gestagen metabolite analysis in the Iberian lynx. To identify faecal gestagen and oestrogen metabolites a radio-metabolism study was performed. Using the progesterone immunoassay two major progesterone metabolites were detected demonstrating that the assay indeed tracks the relevant metabolites. The oestrogen assay measured authentic 17beta-oestradiol and oestrone, and their conjugates. The analysis of the faecal metabolite composition in samples from early and late pregnancy and lactation particularly revealed a distinct shift in the relation between 17beta-oestradiol and oestrone that changed in favour of oestrone. This might indicate different hormone sources during and after pregnancy (corpus luteum, placenta). We hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may led to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

The Culture of Zygotes to the Blastocyst Stage Changes the Postnatal Expression of an Epigentically Labile Allele, Agouti Viable Yellow, in Mice1

Restricting the growth of the embryo can cause adverse whole-of-life changes in an organism's homeostasis. Such adverse long-term consequences may occur even when growth restriction occurs only during the preimplantation period. The molecular basis for these long-term effects has not been defined, although an epigenetic mechanism is suspected. Some loci seem to be more sensitive to epigenetic perturbation than others, and the agouti viable yellow allele (A(vy)) is the best studied example of this. It has active (hypomethylated) and inactive (hypermethylated) epialleles. This study used the A(vy) model to show that growth restriction of preimplantation embryos, as provided by culture of zygotes, induced persistent epigenetic changes that resulted in altered postnatal phenotype. C57BL/6 A(vy)/a males were mated to ovulation-induced FVB/N females, and then either zygotes were collected and cultured for 96 h and the resulting blastocysts were transferred to pseudopregnant recipient females, blastocysts were collected from females and transferred without embryo culture, or pregnancy was allowed to proceed after mating without intervention. Culture was in a commercial human in vitro fertilization media. The proportion of pups expressing the active (hypomethylated) epiallele and yellow coat was significantly higher following zygote culture compared to embryos that were transferred without culture (P = 0.014) or natural matings (P < 0.001). There was no difference in expression of the active epiallele in pups resulting from embryo transfer (without culture) compared to natural matings. These results show for the first time that the preimplantation embryo's growth environment can affect the postnatal expression of a defined epigenetically sensitive allele.

Culture of Mouse Zygotes Induces Epigenetic Changes in the Expression of the agouti viable yellow Allele in the Resulting Progeny.

It is well recognised that perturbation of fetal growth may influence postnatal homeostasis. Recent evidence indicates that the growth environment of the preimplantation embryo may also have long-term consequences for progeny. Epigenetic modification of the genome is one molecular mechanism that could explain a persistence of altered gene expression. This study aimed to assess whether culture of zygotes through to the blastocyst stage, followed by their transfer to recipient females, induced defined epigenetic changes in gene expression that persisted through to adulthood. The agouti viable yellow (Avy) allele is epigenetically sensitive, and is a widely used model for studying epigenetic phenomena. It has active and inactive epialleles that are hypo- and hyper-methylated, respectively. Progeny inheriting the allele in its active state have yellow fur; those with the inactive epiallele have an agouti coat. Some progeny are mosaics for the two epigenetic states and have a mottled coat color. Males (Avy/a C57BL/6) carrying the epiallele were mated to wildtype females (A/A FVB). The following treatments were applied: (1) zygotes were collected and cultured for 94 hours and the resulting blastocysts transferred to recipient females; (2) blastocysts were collected from females and then transferred to recipients without being subjected to embryo culture; and (3) pregnancy was allowed to proceed without intervention. Zygotes were cultured in Sydney IVF Media suite (Cook Womens Health, Australia), a widely used commercial sequential human embryo culture media formulation. Embryos were transferred to day 3 (post coitum) pseudopregnant white Swiss recipient females. The number, sex, genotype, and epi-phenotype of the resulting progeny were recorded. The culture of zygotes caused a significant shift in the expression of the epialleles in progeny compared to embryos that were transferred without culture (P=0.014), or those born following natural matings (P<0.001). Offspring that resulted from embryo culture are more likely to have active (hypomethyalted) epialleles, resulting in a higher proportion of adult progeny with yellow fur. The distribution of epialleles was not different (P>0.05) for the two control treatments not subjected to embryo culture. Embryo culture had no effect on the survival of embryos carrying the Avy allele compared to the wildtype allele. The study shows that culture of mouse zygotes through to the blastocyst stage changed the expected post-natal expression of an epigenetically sensitive allele. Embryo culture has been shown to cause many acute changes to the pattern of gene expression, and there are case reports of human ART progeny with abnormalities with an epigenetic basis. The results of this study provide evidence for the epigenetic basis for the long-term memory of preimplantation embryos growth environment, and for robust and readily accessible methodology for addressing the underlying molecular bases for this phenomenon.

Factors From Damaged Sperm Affect Its DNA Integrity and Its Ability to Promote Embryo Implantation in Mice

Endogenous nucleases in mouse sperm can be activated by freeze-thawing the spermatozoa in media without cryoprotection and cleaving spermatozoa DNA. The role of sperm chromatin integrity during intracytoplasmic sperm injection (ICSI) is of critical importance. We analyzed in the B6D2 mouse the proportion of DNA-fragmented spermatozoa (DFS) produced by incubation in conditioned medium (CM) generated by freeze-thawing sperm in the absence of cryoprotection. We then examined the subsequent development, implantation, and offspring obtained after ICSI with incubated spermatozoa. When fresh sperm cells were incubated for 90 minutes in this CM, a significant increase in the amount of DFS was detected by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay (27% vs 4.5% in fresh sperm). After ICSI of fresh and incubated spermatozoa, embryos were cultured in vitro to either the 2-cell or blastocyst stage before they were transferred into pseudopregnant CD1 females. On day 14, recipients were sacrificed, and implantation rates, estimated as the number of live fetuses plus resorptions, were determined. When ICSI was performed with sperm incubated in CM, no effects on fertilization, embryo cleavage, blastocyst rate, or blastocyst morphology were detected; however, the quality of the embryos was affected because the total implantation rate decreased significantly (P < .05) when 2-cell embryos or blastocysts were transferred. Independently of sperm pretreatment, in vitro cultures significantly affected the percentage of live fetuses present on day 14 of pregnancy. These results demonstrated that there are factors released from fragmented spermatozoa capable of inducing DNA fragmentation in intact sperm that may compromise, to some extent, birth rates after ICSI.

Long-Term Preservation of Mouse Spermatozoa as Frozen Testicular Sections

We previously demonstrated that testicular spermatozoa can be preserved as frozen testicular sections, allowing us to preserve male gametes in less space than conventional methods. However, it remains unclear whether the testicular spermatozoa can be preserved for a long period using this procedure. In this study, we examined the function of testicular spermatozoa preserved as frozen testicular sections for l year at -30 or -80 C. Testicular spermatozoa were successfully retrieved from frozen testicular sections preserved at either -30 or -80 C, and their function was assessed using intracytoplasmic sperm injection (ICSI). Over 90% of the oocytes injected with long-term preserved testicular spermatozoa formed pronuclei, which was a frequency similar to that obtained with spermatozoa preserved for a short term, indicating that the testicular spermatozoa retained oocyte activation factor(s). Approximately 70% of the fertilized oocytes developed to 2-cell stage embryos, and 9.3 to 12.8% of the embryos developed to term after transfer into pseudopregnant females, regardless of the preservation temperatures examined. These results indicate that the birthrates of progeny did not differ between the preservation temperatures examined. They also indicate that male gametes can be preserved in testicular frozen sections for at least 1 year without loss of function.

Rat Embryonic Stem-Like (ES-Like) Cells Can Contribute to Extraembryonic TissuesIn Vivo

Despite significant advances achieved through gene targeting in mouse embryonic stem (ES) cells, this technology is presently only available in mice. Because the rat is a species of undeniable importance to biomedical research, attempts at derivation of rat ES cell lines have been ongoing for many years; however, the putative rat ES cell lines that have been reported to date have not yet displayed the ability to contribute in vivo to developing tissues following embryo injection. In contrast to previous studies, we describe herein the successful derivation and characterization of rat ES-like cell lines that not only express markers of undifferentiated cells, alkaline phosphatase (AP) activity and stage-specific embryonic antigen-1 (SSEA-1) cell surface antigen, but also retain expression of Oct4 (also known as Pou5f1) a homeodomain transcription factor and molecular marker of pluripotent cells. Notably, these rat ES-like cells, when injected into blastocysts transferred to pseudopregnant females, can contribute to developing extraembryonic tissues. This report demonstrates for the first time that rat ES-like cells can be derived efficiently, can express a panel of pluripotent cell markers, can be genetically modified in vitro and cryopreserved, and importantly, are capable of contributing to extraembryonic tissues in vivo.

Mechanisms of Action of Regulatory T Cells Specific for Paternal Antigens During Pregnancy

To investigate whether pregnancy-induced regulatory T cells are generated specifically for paternal antigens or expanded by hormonal changes and to study regulatory T cell-related mechanisms during pregnancy. We used murine models of normal, abortion-prone, and pseudopregnancy to characterize regulatory T cells and hormones by methods such as flow cytometry, molecular biology techniques, and chemiluminescence. Antigen specificity was studied in experiments in which animals were vaccinated with paternal antigens or adoptively transferred with regulatory T cells. To analyze regulatory T cell-mediated mechanisms, we used neutralizing antibodies against IL-10 or TGF-beta. Regulatory T cells are activated by male antigens, and minor antigens are protected by linked immunosuppression. Our data exclude the possibility that regulatory T cell expansion during pregnancy is exclusively driven by hormonal changes. An increase in systemic regulatory T cell levels in pseudopregnant females after mating with vasectomized males but not after pseudopregnancy induced mechanically confirms generation of regulatory T cells specific for paternal antigens. As for the mechanisms, neutralizing IL-10 abrogates the protective effect of regulatory T cells, whereas blockage of TGF-beta does not provide the same effect. Our data confirm that regulatory T cells act in an antigen-specific manner during pregnancy and strongly suggest that IL-10 is involved in regulatory T cell-mediated protection of the fetus. These data contribute to the knowledge of the basic mechanisms regulating immune tolerance during pregnancy, a major biologic question with important medical implications. II.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Mitochondrial Lipid Metabolism and Function Resulting in a Deficient Mitochondrial Bioenergetic Phenotype

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Transposon-Mediated Transgenesis in Rats

INTRODUCTIONThis protocol describes a method for the production of transgenic rats by coinjecting circular PiggyBac transposon-containing plasmid DNA with transposase-encoding mRNA. After fertilized eggs are collected from females, pronuclei are microinjected with the DNA and mRNA and then transferred immediately into the oviducts of pseudopregnant foster females. Using this procedure, we have obtained transformation frequencies of 33%-100% (average of ~80%). Traditional methods for generating transgenic rats (i.e., microinjection of linear DNA) yield 3%-41% transgenic founders. Thus, transposon-mediated transformation of fertilized rat eggs is an efficient alternative method to generate transgenic rats.

PRESERVATION OF EJACULATED MOUSE SPERMATOZOA FROM FERTILE C57BL/6 AND INFERTILE Hook1/Hook1 MICE COLLECTED FROM THE UTERI OF MATED FEMALES

Methods routinely used to preserve mouse spermatozoa require that the male be euthanized to recover spermatozoa from the epididymides. Here we obtained multiple samples of ejaculated spermatozoa from normal fertile C57BL/6 and infertile Hook1/Hook1 (formerly known as azh/azh) mutant males from uteri after mating, thus avoiding terminating the males. Ejaculated sperm were preserved by conventional cryopreservation or by rapid freezing without cryoprotection, and injected into the oocytes by intracytoplasmic sperm injection (ICSI). The proportions of oocytes that survived, became activated, and developed into two-cell embryos were similar when comparing two preservation methods, wild type vs. Hook1/Hook1 mice, and tested mice vs. controls (fresh and rapid frozen epididymal and fresh ejaculated sperm). Two-cell embryos were transferred into the oviducts of pseudo-pregnant females and fetal development was examined at day 15 of gestation. Thirty nine to 54% of transferred embryos produced with preserved ejaculated sperm implanted. Live, normal fetuses (11–17%) were obtained in all examined groups and from all males included in the study. More implants (71–82%) and fetuses (28–31%) were noted in controls. Lower developmental potentials of embryos produced with preserved ejaculated sperm might be due to their capacitation status; the majority of sperm retrieved from the uterus were capacitated. This study bears significance for the maintenance and distribution of novel mouse strains. The method is applicable for all types of mice, including those with male infertility syndromes. The sole requirement is that the male of interest is able to copulate and its ejaculate contains spermatozoa. [This material is based on work supported by NIH HD048446 grant to M.A.W] (platform)

Keratinocyte-specific Expression of Fatty Acid Transport Protein 4 Rescues the Wrinkle-free Phenotype in Slc27a4/Fatp4 Mutant Mice

FATP4 (fatty acid transport protein 4; also known as SLC27A4) is the most widely expressed member of a family of six long chain fatty acid transporters. FATP4 is highly expressed in enterocytes and has therefore been proposed to be a major importer of dietary fatty acids. Two independent mutations in Fatp4 cause mice to be born with thick, tight, shiny, "wrinkle-free" skin and a defective skin barrier; they die within hours of birth from dehydration and restricted movements. In contrast, induced keratinocyte-specific deficiency of FATP4 in adult mice causes only mild skin abnormalities. Therefore, whether the loss of FATP4 from skin or a systemic gestational metabolic defect causes the severe skin defects and neonatal lethality remain important unanswered questions. To investigate the basis for the phenotype, we first generated wild-type tetraploid/mutant diploid aggregates that should lead to rescue of any abnormalities caused by loss of FATP4 from the placenta. However, the skin phenotype was not ameliorated. We then generated transgenic mice expressing exogenous FATP4 either widely or specifically in suprabasal keratinocytes, and we bred the transgenes onto the Fatp4(-/-) background. Both modes of FATP4 expression led to rescue of the neonatally lethal skin defects, and the resulting mice were viable and fertile. Keratinocyte expression of an FATP4 variant with mutations in the acyl-CoA synthetase domain did not provide any degree of rescue. We conclude that expression of FATP4 with an intact acyl-CoA synthetase domain in suprabasal keratinocytes is necessary for normal skin development and that FATP4 functions in establishing the cornified envelope.