Live pups from evaporatively dried mouse sperm stored at ambient temperature for up to 2 years.

The purpose of this study is to develop a mouse sperm preservation method based on evaporative drying. Mouse sperm were evaporatively dried and stored at 4°C and ambient temperature for 3 months to 2 years. Upon rehydration, a single sperm was injected into a mature oocyte to develop into a blastocyst after culture or a live birth after embryo transfer to a recipient female. For the samples stored at 4°C for 3, 6, 12, 18, and 24 months, the blastocyst formation rate was 61.5%, 49.1%, 31.5%, 32.2%, and 41.4%, respectively. The blastocyst rate for those stored at ambient temperature (∼22°C) for 3, 6, 12, and 18 months was 57.8%, 36.2%, 33.6%, and 34.4%, respectively. Fifteen, eight and three live pups were produced from sperm stored at room temperature for 12, 18, and 24 months, respectively. This is the first report of live offspring produced from dried mouse sperm stored at ambient temperature for up to 2 years. Based on these results, we suggest that evaporative drying is a potentially useful method for the routine preservation of mouse sperm.

Preservation of mouse sperm by convective drying and storing in 3-O-methyl-D-glucose.

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.

Mouse sperm desiccated and stored in trehalose medium without freezing.

Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4 degrees C and 22 degrees C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22 degrees C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4 degrees C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22 degrees C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4 degrees C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.

The cell cycle associated protein, HTm4, is expressed in differentiating cells of the hematopoietic and central nervous system in mice.

HTm4 is a member of a newly defined family of human and murine proteins, the MS4 (membrane-spanning four) protein group, which has a distinctive four-transmembrane structure. MS4 protein functions include roles as cell surface signaling receptors and intracellular adapter proteins. We have previously demonstrated that HTm4 regulates the function of the KAP phosphatase, a key regulator of cell cycle progression. In humans, the expression of HTm4 is largely restricted to cells of the hematopoietic lineage, possibly reflecting a causal role for this molecule in differentiation/proliferation of hematopoietic lineage cells. In this study, we show that, like the human homologue, murine HTm4 is also predominantly a hematopoietic protein with distinctive expression patterns in developing murine embryos and in adult animals. In addition, we observed that murine HTm4 is highly expressed in the developing and adult murine nervous system, suggesting a previously unrecognized role in central and peripheral nervous system development.

Intracytoplasmic sperm injection (ICSI) enables rescue of valuable mutant mouse strains.

Genetically altered mice are important research tools for the study of human development and disease. Occasionally, whether or not related to the genetic mutation, mice may become infertile with age and, thus, risk loss of the mutant line. Under conditions in which assisted reproduction techniques (ARTs), such as in vitro fertilization, are unsuccessful, a new strategy, intracytoplasmic sperm injection (ICSI), may be applicable. This technique has been perfected for use in the mouse and is now considered a reliable, effective, and efficient ART. In the study reported here, we "rescued" (i.e., produced offspring, using ICSI from a "last-of-line" mutant male mouse) four lines that otherwise had become infertile and unresponsive to conventional ART's. A total of 26 live pups were produced from eight pregnant recipient foster mothers. Five mutant male mice were derived (one each from three lines, and two from one line), and all survived to adulthood. We found that live born mice could be successfully derived by use of ICSI that subsequently could breed by natural mating to reestablish the mutant line. Because of its effectiveness and reliability under these conditions, ICSI should be considered a powerful addition to the armamentarium of ART's applicable in the genetically-altered mouse, especially when only one male may still be available.

Quantitative microinjection of trehalose into mouse oocytes and zygotes, and its effect on development.

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.

Nkx2.5 homeoprotein regulates expression of gap junction protein connexin 43 and sarcomere organization in postnatal cardiomyocytes.

Nkx2.5, an evolutionarily conserved homeodomain containing transcription factor, is one of the earliest cardiogenic markers. Although its expression continues through adulthood, its function in adult cardiomyocytes is not well understood. To examine the effect of Nkx2.5 in terminal differentiated postnatal cardiomyocytes, we generated transgenic mice expressing either wild-type Nkx2.5 (TG-wild), a putative transcriptionally active mutant (carboxyl-terminus deletion mutant: TG-DeltaC) or a DNA non-binding point mutant of Nkx2.5 (TG-I183P) under alpha-myosin heavy chain promoter. Most TG-wild and TG-DeltaC mice died before 4 months of age with heart failure associated with conduction abnormalities. Cardiomyocytes expressing wild-type Nkx2.5 or a putative transcriptionally active mutant (DeltaC) had dramatically reduced expression of connexin 43 and changed sarcomere structure. Wild-type Nkx2.5 adenovirus-infected adult cardiomyocytes demonstrated connexin 43 downregulation as early as 16 h after infection, indicating that connexin 43 downregulation is due to Nkx2.5 overexpression but not due to heart failure phenotype in vivo. These studies indicate that overexpression of Nkx2.5 in terminally differentiated cardiomyocytes dramatically alters cardiac cell structure and function.

Desiccation tolerance of spermatozoa dried at ambient temperature: production of fetal mice.

Long-term preservation of mouse sperm by desiccation is economically and logistically attractive. The current investigation is a feasibility study of the preservation of mouse sperm by convective drying in an inert gas (nitrogen). Mouse sperm from the B6D2F1 strain isolated in an EGTA-supplemented Tris-HCl buffer were dried using three different drying rates and were stored for 18-24 h at 4 degrees C. The mean final moisture content was <5% for all the protocols. After intracytoplasmic sperm injection (ICSI), the mean blastocyst formation rates were 64%, 58%, and 35% using the rapid-, moderate-, and slow-drying protocols, respectively. The slow-drying protocol resulted in a rate of development significantly lower than that observed using rapid- and moderate-drying protocols and indicated that a slower drying rate may be detrimental to the DNA integrity of mouse sperm. The transfer of 85 two- or four-cell embryos that were produced using rapidly desiccated sperm resulted in 11 fetuses (13%) on Day 15 compared with the production of 34 fetuses (40%) produced using the transfer of 86 two- or four-cell embryos that were produced using fresh sperm (P < 0.05). The results demonstrate the feasibility of using a convective drying protocol for the successful desiccation of mouse sperm and identifies some of the important parameters required for optimization of the procedure.

Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A(164), we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A-expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A(164) also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new "giant" lymphatics generated by VEGF-A(164) were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.