Cross fertilization in vivo and in vitro between three species of vesper mice, Calomys (Rodentia, Cricetidae)

Cross fertilization was tested between oocytes of Calomys callidus and spermatozoa from C. callidus, C. musculinus and C. laucha by both in vivo and in vitro insemination. After in vivo and in vitro insemination, respectively, percentages of oocytes fertilized were 68.8 and 46.6 (C. callidus X C. callidus), 20.3 and 12.7 (C. callidus X C. musculinus), 26.7 and 4.3 (C. callidus X C. laucha). Thus, the percentages obtained after in vitro insemination were always lower than those obtained with in vivo insemination. It was found that 23.9% and 44.4% of two-cell hybrid embryos were present in oviducts 30 hr after in vivo insemination of C. callidus females with C. musculinus or C. laucha spermatozoa, respectively. At a later stage (56 hr postinsemination), development did not progress further, and abnormal embryos were found both at 30 and 56 hr postinsemination, suggesting some kind of cleavage arrest or degeneration of the embryos. We suggest that fertilization is not strictly species-specific, at least among the species we studied, but that there are some factors that reduce the efficiency of interspecific fertilization.     

Successful capacitation and homologous fertilization in vitro in Calomys musculinus and Calomys laucha (Rodentia - sigmodontinae)

Small South American rodents of the genus Calomys have been used extensively for virology and ecological research. Previous studies have demonstrated that Calomys musculinus and Calomys laucha have a relatively short oestrous cycle and that superovulation and parthenogenetic activation can be induced. The purpose of this study was to determine the requirements for in vitro manipulation of the male gamete and in vitro fertilization. Two culture media and different concentrations of spermatozoa were tested for their ability to support sperm motility, hyperactivation and the acrosome reaction. The ability of capacitated Calomys spermatozoa to penetrate zona-free hamster eggs was also evaluated. In vitro fertilization was assessed by examining attachment and binding to the zona pellucida, second polar body extrusion, pronucleus formation and the fertilizing sperm tail. The results of the study showed that: (i) Tyrode's albumin lactate pyruvate (TALP) medium was more effective than T6 medium for maintaining sperm motility in vitro; (ii) hyperactivation was achieved with TALP but not with T6; (iii) the acrosome reaction was easily distinguished by light microscopy and depends on time and sperm concentration; (iv) capacitated spermatozoa are able to penetrate zona-free hamster eggs; and (v) superovulated oocytes can be fertilized in vitro. This is the first report of capacitation and in vitro fertilization for Calomys sp. These results provide opportunities to use C. musculinus and C. laucha as new laboratory animals for research into reproductive biology.     

Superovulation in vesper mice, Calomys laucha--an important biomedical model for hantavirus and arenavirus (Rodentia-Sigmodontinae)

Sigmodontine rodents are poorly studied and have not received much attention as a reproductive model. Renewed interest in the South American rodents has been stimulated by their link to endemic diseases that are transmitted to man. Calomys laucha acts as a reservoir of two dangerous viruses: an arenavirus named 'Junin virus', the aetiological agent of Argentinian haemorrhagic fever, and the hantavirus, both of which constitute serious sanitary problems. The aim of this study was to establish suitable conditions to superovulate the vesper mouse, Calomys laucha. We examined the hormonal doses, the time interval between hormones, the time-course of ovulation, and the effect of female age on the response to exogenous hormone administration. Female mice were injected with 5-5, 8-8 or 12-15 IU of PMSG/hCG, 48 h apart, at different age intervals (from 30 to > 120 days old). The best superovulation rate was obtained with 8-8 IU PMSG/hCG. Ovulation started about 10 h post-hCG and was completed during the next 4-5 h, and was achieved irrespectively from the oestrus cycle stage. The number of oocytes was influenced by the age of the females. The youngest females had only a superovulatory response. Females older than 61 days showed both ovulatory and superovulatory responses, although 91-120-day-old females had a high ovulatory response. Most of the oocytes (96.5%) recovered were morphologically normal. The genus Calomys constitutes a reproductive model completely different from conventional laboratory rodents.     

A new species of Syphacia (Nematoda: Oxyuridae) from Calomys laucha (Rodentia: Cricetidae) in an agroecosystem of central Argentina

A new oxyurid nematode Syphacia hodarae n. sp. is described from the cecum and rectum of the cricetid rodent Calomys laucha Fischer, 1814 (Sigmodontinae, Phyllotini), captured in an agroecosystem of central Argentina. The new species is distinguished from other members of the genus mainly by the shape of the cephalic plate, presence of cervical alae in females, absence of lateral alae, and absence of deirids. Some characters are shared with Syphacia carlitosi, a parasite of Akodon azarae from the wetlands in Argentina. However, S. hodarae can be differentiated from this species by the absence of ornamentation on the accessory hook of the gubernaculum, length of spicule and gubernaculum, size of the eggs, and distance to the vulva from the anterior end. This is the first record of a Syphacia species from the tribe Phyllotini in Argentina, and the first time a Syphacia species is reported from C. laucha .     

Calomys laucha (Rodentia, Cricetidae): growth and breeding in laboratory conditions

The husbandry and breeding of Calomys laucha (Rodentia, Cricetidae) in captivity are described. Growth curves based on body weight and length showed statistical differences between sexes after 45 days, males being heavier than females. The overall reproductive efficiency was 53.4% but birth rate was depressed during winter. Gestation length was 21 +/- 1 days and females exhibited postpartum oestrus with a 3-7 day implantation delay (51%). Litter size was 5.3 +/- 1.1 (n = 34). Pup survival at weaning was 84.9%. Mean life span in laboratory conditions was 13.5 months and a cumulative mortality of 90% was reached at 27-28 months of age.     

Density-dependent habitat selection between maize cropfields and their borders in two rodent species (Akodon azarae and Calomys laucha) of Pampean agroecosystems

We studied habitat preferences and intra and interspecific density-dependent effects on habitat selection by Akodon azarae and Calomys laucha between maize fields and their adjacent borders, during different developmental stages of the crop. Akodon azarae detected quantitative differences between habitats, using preferentially borders throughout the year, while C. laucha perceived borders and cropfields as quantitatively similar during spring and summer and it detected borders as quantitatively better at the high density period (autumn and winter). These results support the prediction of differential habitat preferences as a model of community organisation at the low density period, while they are consistent with shared habitat preferences during autumn and winter when both species apparently coexist in the better habitat (border). Akodon azarae showed intraspecific density-dependent habitat selection throughout the year, except in spring, while habitat selection by C. laucha was density-dependent in spring, autumn and winter. The effect of interspecific density on habitat selection was detected in both habitats and changed seasonally. The effect of A. azarae over C. laucha by resources exploitation was detected in borders, while competitive effects of C. laucha over A. azarae was observed within cropfields. Both species were more affected by exploitation competition than interference, which was more common in borders than in maize fields. We conclude that seasonally have a profound effect in habitat selection of these species because it changes the intensity of intra and interspecific competition and affects different habitat preferences and basic suitability of habitats. This revised version was published online in November 2006 with corrections to the Cover Date.     

In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm

Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.     

In vitro embryo production in buffalo (Bubalus bubalis) using sexed sperm and oocytes from ovum pick up

The objective was to explore the use of sexed sperm and OPU-derived oocytes in an IVP system to produce sex-preselected bubaline embryos. Oocytes were recovered from 20 fertile Murrah and Nili-Ravi buffalo cows by repeated (twice weekly) ultrasound-guided transvaginal ovum pick up (OPU), or by aspiration of abbatoir-derived bubaline ovaries, and subjected to IVF, using frozen-thawed sexed or unsexed bubaline semen. On average, 4.6 oocytes were retrieved per buffalo per session (70.9% were Grades A or B). Following IVF with sexed sperm, oocytes derived from OPU had similar developmental competence as those from abattoir-derived ovaries, in terms of cleavage rate (57.6 vs. 50.4%, P=0.357) and blastocyst development rate (16.0 vs. 23.9%, P=0.237). Furthermore, using frozen-thawed sexed versus unsexed semen did not affect rates of cleavage (50.5 vs. 50.9%, P=0.978) or blastocyst development (15.3 vs. 19.1%, P=0.291) after IVF using OPU-derived oocytes. Of the embryos produced in an OPU-IVP system, 9 of 34 sexed fresh embryos (26.5%) and 5 of 43 sexed frozen embryos (11.6%) transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos (26.9%) and 6 out of 39 unsexed frozen embryos (15.4%) transferred to recipients established pregnancies. Eleven sex-preselected buffalo calves (10 females and one male) and 10 sexed buffalo calves (six females and four males) were born following embryo transfer. In the present study, OPU, sperm sexing technology, IVP, and embryo transfer, were used to produce sex-preselected buffalo calves. This study provided proof of concept for further research and wider field application of these technologies in buffalo.     

In vitro heterologous cytotoxicity by T effector cells from mice immunized with Sindbis virus.

An in vitro correlate of cell-mediated cross-protection among alpha-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.     

In vitro and cellular assays for palmitoyl acyltransferases using fluorescent lipidated peptides

Protein palmitoylation is emerging as an important post-translational modification in development as well as in the establishment and progression of diseases such as cancer. This chapter describes the use of fluorescent lipidated peptides to characterize palmitoyl acyltransferase (PAT) activities in vitro and in intact cells. The peptides mimic two motifs that are enzymatically palmitoylated, i.e. C-terminal farnesyl and N-terminal myristoyl sequences. These substrate peptides can be separated from the palmitoylated product peptides by reversed-phase HPLC, detected and quantified by the fluorescence of their NBD label. Through these methods, the activities of PATs toward these alternate substrates in isolated membranes or intact cells can be quantified. The in vitro assay has been used to characterize human PATs and to identify inhibitors of these enzymes. The cellular assay has been useful in elucidating the kinetics of protein palmitoylation by PATs in situ, and the sub-cellular distribution of the palmitoylated products.     

In vitro transformation assays for chemical carcinogens

A variety of in vitro mammalian cell assays, designed specifically for the identification of carcinogenic compounds, have been in operation for more than a decade. Although no individual transformation system has won universal acceptance during this time, recent advances have led to the improved reliability and sensitivity of a number of these short-term tests. The underlying problems associated with the most widely used assays are identified and new developments in this rapidly expanding field are noted and discussed.     

In vitro feeding assays for hard ticks

Prevention of tick bites and transmission of tick-borne pathogens requires the use of molecules that target physiological processes crucial to both tick and pathogen survival. These molecules are best tested in standardized in vitro assays. Because hard ticks require several days to feed to repletion, the development of in vitro feeding assays for these species is challenging. A standard and easily automated feeding assay has been developed for the tick Ixodes ricinus that involves feeding on blood through a membrane that mimics the elasticity of skin. The system can be adapted to feed other hard tick species in vitro. This assay permits, among others, investigations on the role of tick endosymbionts on tick survival, the identification of potential vaccine candidates and drugs, and the application of genomic tools in vitro, including RNA interference experiments.     

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10⁶ sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.     

Developing an improved In vitro propagation system for slow-growing species using garcinia mangostana L. (mangosteen)

Summary This investigation disclosed that evaluation of tissue culture parameters of slowly developing species (e.g. Garcinia mangostana) requires monitoring of treatments through two or more successive, relatively long passages. Two 8-wk passages were necessary to observe differences in phytohormone effects. Photoperiod and temperature effects were not clearly evident until tissues had been cultured through three passages; the optimal photoperiod and temperature for shoot proliferation could not be established until after the fifth passage. Our investigation revealed that no auxin supplementation was necessary for bud primordium differentiation in cotyledon explants or proliferation of regenerated shoots. The optimum N⁶-benzyladenine concentration for primordium differentiation was 13.3 μM, and for shoot proliferation ranged from 4.4 to 13.3 μM. Continuous culturing in an 8-h photoperiod at 30°C resulted in progressively intensified degeneration of shoots after three passages. In contrast, successive passages in a 16-h photoperiod/26°C regimen enabled sustained regeneration of shoots. The shoots rooted at a rate of 85% when precultured for 3 d in a medium containing 4921.3 μM indole-3-butyric acid, or 10 d at 492.1 μM, then cultured for two 8-wk passages in phytohormone-free medium. Following acclimatization by gradually lowering the relative humidity in the growth chamber, rooted shoots survived transfer to the greenhouse at a rate of 95%.     

In vivo and in vitro protein solubility assays using split GFP

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.     

In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces

The first event occurring at the boundary between a metal implant and living tissue is the attachment of cells onto the metal surface of the implant. The attachment characteristics of the metal in this situation are critical in determining its biocompatibility and usefulness as artificial bone and tooth implants. Using the human osteosarcoma cell line Saos-2, we attempted to establish simple and reliable methods for evaluating the attachment of cultured osteoblastic cells onto titanium samples that had been subjected to various surface treatments. Fluorescence actin imaging showed that cells cultured on titanium with hydrofluoric acid etching (HF-Ti) exhibited delayed spreading of their cytoplasm, as compared to cells cultured for the same length of time on nitrided titanium or physically polished titanium. The HF-Ti-cultured cells also exhibited poor assembly of focal contacts, as visualized by vinculin immunofluorescence. Furthermore, in motility assays based on an in vitro wound model, cells cultured on HF-Ti migrated more slowly than cells cultured on other titanium surfaces. These data suggest that Saos-2 cells attach less effectively to the HF-Ti surface. The methods described in this study should be useful for assessing the initial interactions of cultured cells with various materials, including metals.S.G. is the recipient of a grant awarded to foreign students by the government of Japan. This study was supported by the Integrated Center for Science (INCS) at Ehime University.     

A method for hormonal induction of sperm release in anurans (eight species) and in vitro fertilization in Lepidobatrachus species

Injections of synthetic human gonadotropin releasing hormone (GnRH) into the dorsal pelvic area were used in an attempt to stimulate sperm release in isolated males of eight anuran species including Xenopus laevis, Rana pipiens and Lepidobatrachus laevis . Sperm were obtained within 1–5 h post injection either by mechanical stimulation or by cloacal lavage. Sperm suspensions varied from 8 μL to 7 mL and the cell densities ranged from 4 × 10⁵ to 4 × 10⁷ sperm/mL. The sperm obtained from seven species using GnRH-induced release were viable based on light microscopic observations of motility. In addition, sperm preparations fertilized eggs in vitro and produced normal tadpoles in the case of L. laevis and L. llanensis . This hormonal method of anuran sperm collection will provide a convenient non-injurious way to obtain anuran sperm for basic studies of reproduction and development.