Mouse ICSI with frozen-thawed sperm: the impact of sperm freezing procedure and sperm donor strain.

Normal mouse offspring can be obtained from oocytes injected with frozen-thawed spermatozoa without cryoprotection, however, embryo development can be affected by sperm freezing procedure and sperm donor strain. In this study we observed that direct contact of mouse spermatozoa with liquid nitrogen did not affect their ability to activate injected oocytes but severely restricted subsequent in vitro embryo development to blastocyst stage. Tris-EDTA buffer and M2 were also shown to be better sperm freezing extenders than DPBS, allowing higher developmental potential.

Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo.

The objective of this study was to examine the time during the postfertilization period that gene expression patterns in in vitro-cultured bovine embryos diverge from those of their in vivo-cultured counterparts. Presumptive bovine zygotes were produced by in vitro maturation and fertilization of immature oocytes collected from the ovaries of slaughtered animals. Approximately 20 h post insemination (hpi), zygotes were denuded and randomly divided into two groups for culture either in vitro, in synthetic oviduct fluid medium, or in vivo, in the ewe oviduct.

Hyperglycemia-induced apoptosis affects sex ratio of bovine and murine preimplantation embryos.

The effect of glucose in the medium used during in vitro culture on both cell death by apoptosis and the sex ratio of bovine blastocysts derived from in vitro-matured and in vitro-fertilized oocytes was evaluated. Oocytes were matured, inseminated, and cultured in vitro in mSOF medium with 10% FCS with or without glucose supplementation. Exposure to high concentrations of glucose (10, 20, and 30 mM) during bovine embryo development in vitro from zygote to blastocyst resulted in a decrease in the number of cells per embryo and an increase in the frequency of apoptotic cells.

Early detection of PrPres in BSE-infected bovine PrP transgenic mice.

Transgenic mouse lines expressing different levels of the bovine prion protein gene (boPrP(C)) were generated. Upon infection with BSE prions, all transgenic lines tested exhibited characteristics of the bovine disease. Typical CNS spongiform degeneration was observed by histopathology and presence of PrP(res) could be detected both by Western blot and immunohistochemistry (IHC) assays, confirming for this model the absence of an interspecies barrier to BSE infection.

Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression.

We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns.

Expression of the FUS domain restores liposarcoma development in CHOP transgenic mice.

Fusion proteins created by chromosomal abnormalities are key components of mesenchymal cancer development. The most common chromosomal translocation in liposarcomas, t(12;16)(q13;p11), creates the FUS-CHOP fusion gene. In the past, we generated FUS-CHOP and CHOP transgenic mice and have shown that while FUS-CHOP transgenic develop liposarcomas, mice expressing CHOP, which lacks the FUS domain, display essentially normal white adipose tissue (WAT) development, suggesting that the FUS domain of FUS-CHOP plays a specific and critical role in the pathogenesis of liposarcoma.

Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality.

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded.

Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro.

The effect of glucose in the medium used during in vitro culture on the sex ratio of bovine blastocysts derived from in-vitro-matured and in-vitro-fertilized oocytes was evaluated. Oocytes were matured and inseminated with mixed sperm from three bulls and were cultured in vitro in modified synthetic oviducal fluid medium with 10% fetal calf serum, with or without glucose supplementation. The overall rate of cleaved embryos that developed to expanded blastocyst in the medium without glucose (27.

CMV-driven expression of green fluorescent protein (GFP) in male germ cells of transgenic mice and its effect on fertility.

To determine if the expression of green fluorescent protein (GFP) during spermatogenesis can compromise the fertility of transgenic animals, we have produced mouse transgenic lines expressing GFP in the testis under the control of the potent immediate early promoter and enhancer region of the human cytomegalovirus (CMV). GFP expression was detected in the germ cells with no apparent effect on the fertility of any of the five transgenic lines studied. We demonstrate that the CMV promoter is transcriptionally active in the testes of mice aged 7 days.

Hematopoietic stem cell transplantation in utero produces sheep-goat chimeras.

Both allogeneic and xenogeneic hematopoietic chimera models have been developed, including fetal sheep models that demonstrated high levels of stable, multilineage engraftment created by in utero hematopoietic stem cell transplantation. The aim of this study was to test the efficacy of in utero transplantation to create xenogeneic sheep-goat hematopoietic chimeras. Fetal liver cells and T-cell-depleted adult bone marrow were tested as sources of hematopoietic stem cells.

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos.

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments.

Demographic and behavioral determinants of the reduction of male-to-female birth ratio in Spain from 1981 to 1997.

The ratio of male-to-female births has been declining in Spain since 1981. In the last few decades, the proportion of male newborns has also been decreasing in other industrialized countries. It has been hypothesized that these declines are due to environmental factors such as a longer exposure to environmental pollutants, hormonal levels, or sexual behavior.

Relationship between time of first cleavage and the expression of IGF-I growth factor, its receptor, and two housekeeping genes in bovine two-cell embryos and blastocysts produced in vitro.

We have previously demonstrated that there is a clear relationship between the time interval between insemination and first cleavage in vitro and the development to the blastocyst stage of bovine embryos. In addition we have shown that this developmental ability can be linked to the stability of the mRNA for several gene transcripts measured in 2-cell bovine embryos cleaving at different times. The aim of this study was to examine the relationship between bovine embryo developmental competence, assessed in terms of time of first cleavage, and the expression of insulin-like growth factor-I (IGF-I) ligand and receptor, hypoxanthine phosphoribosyl transferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD).

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos.

The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin.

The chimeric FUS/TLS-CHOP fusion protein specifically induces liposarcomas in transgenic mice.

The characteristic t(12;16)(q13;p11) chromosomal translocation, which leads to gene fusion that encodes the FUS-CHOP chimeric protein, is associated with human liposarcomas. The altered expression of FUS-CHOP has been implicated in a characteristic subgroup of human liposarcomas. We have introduced the FUS-CHOP transgene into the mouse genome in which the expression of the transgene is successfully driven by the elongation factor 1alpha (EF1alpha) promoter to all tissues.

Superovulatory response of Murciana goats to treatments based on PMSG/Anti-PMSG or combined FSH/PMSG administration.

Superovulation in goats is frequently restricted by the cost of gonadotropin or the handling requirements. In this situation PMSG has the advantage of a lower cost and single dose protocol, but the variability of response obtained restricts its use. Thus, 2 alternative treatments with the advantages of PMSG were tested.

Transgenesis in large domestic species: future development for milk modification.

The present review has two goals. First, to offer an overview of recent advances in the technical strategies applied to the production of transgenic large domestic animals, and second, to review how transgenic technology can be applied to the modification of milk composition. Transgenic sheep and cattle obtained through nuclear transfer are now a reality, opening up a means of ruminant transgenic production with an efficiency that entitles us to consider it a serious alternative to microinjection.

Differential expression of two genes located on the X chromosome between male and female in vitro-produced bovine embryos at the blastocyst stage.

The potentially unbalanced expression at preimplantation developmental stages of X-linked genes might be responsible of the faster development of male than female embryos in vitro. Two genes located on the X chromosome, glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT), are involved in controlling the amount of oxygen radicals, and hence they might have influence in embryo development. We have quantified mRNA expression of these two genes, using in vitro fertilized-in vitro cultured male and female bovine embryos.

Effect of post-thaw cell rehydration at 4 degrees C on survival of frozen and vitrified IVP-derived bovine embryos.

In vitro-produced bovine embryos (IVP) were either frozen in 10% glycerol in a phosphate-buffered saline solution (PBS) using conventional slow freezing or vitrified in 25% glycerol and 25% ethylene glycol in PBS. The results of viability and hatching rates were compared between frozen and vitrified embryos after thawing and dilution using one of three different protocols: (A) a three-step dilution procedure, (B) a one-step dilution procedure or (C) a procedure in which embryos were kept in situ inside the straw at 4 degrees C for 10 min during a one-step dilution procedure. No significant differences in embryo survival were found among protocols A, B and C for frozen embryos and between protocols A and B for vitrified embryos.

Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte.

The aim of this study was to explore how some reproductive methodologies may affect the sex ratio. We first confirmed the association between the maturation stage of bovine oocytes at the time of in vitro fertilisation (IVF) and the sex ratio of in vitro-derived embryos. Secondly, we studied whether the time of insemination, prior to or after ovulation, could alter the sex ratio in sheep.

Isolation of pluripotent stem cells from cultured porcine primordial germ cells.

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). To date, EG cells have been isolated only in the mouse. Murine EG cells share several characteristics with embryonic stem (ES) cells, including morphology, pluripotency, and the capacity for germline transmission.

Early transcription of the SRY gene by bovine preimplantation embryos.

We have examined mRNA expression of two genes located on the Y chromosome, the sex-determining region Y gene (SRY) and the linked zinc finger gene (ZFY), using in vitro fertilized-in vitro cultured bovine embryos. Expression of the SRY gene, implicated in sex determination in mammals, has been reported to occur both for a short time at the sex-determining stage of development around the period of the primitive undifferentiated gonad and in the adult testis. In this study, using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we detected SRY but not ZFY mRNA expression as early as the 4- to 8-cell stage and through to the blastocyst stage in bovine embryos.

Ovine-specific Y-chromosome RAPD-SCAR marker for embryo sexing.

An accurate, sensitive, and quick (approximately 3 h) method for determining the sex of ovine embryos was developed using polymerase chain reaction (PCR) primers derived from an ovine-specific Y-chromosome random amplified polymorphic DNA marker (UcdO43). The accuracy and sensitivity of the assay were first tested using genomic DNA from 10 males and 10 females of five different sheep breeds, and then tested using serial dilutions of male-in-female DNA. The assay was 100% accurate in confirming the sex of the individuals and the ovine male-specific fragment was detected in dilutions containing as little as 10 pg of male DNA in 50 ng of female DNA.

Relationship between stage of development and sex of bovine IVM-IVF embryos cultured in vitro versus in the sheep oviduct.

We have confirmed more rapid development of male compared with female in vitro-cultured bovine embryos during the first 7 d after in vitro fertilization. The male-to-female sex ratio of expanded blastocysts after 10 d of in vitro culture was 1.37:1.