Improved felid embryo development by group culture is maintained with heterospecific companions

Domestic cat embryos of excellent quality appear to improve development of conspecific embryos when cultured together, providing an avenue for improving development of embryos from valuable species or individuals. To have relevance to rare species, it would be useful to understand if this advantage could be conferred by heterospecific companions because there usually are severely limited numbers of conspecific embryos available from wildlife donors. In the first study, we incubated single test cat embryos alone (controls) or with 10 cat embryos or 10 or 20 mouse embryos under similar regimented conditions (each group shared 20 microl medium). In the second study, single test cat embryos were cultured alone, with 10 conspecific or 20 mouse embryos or 10 cattle embryos (each group shared 20 microl medium). Single test embryos in all treatment groups achieved similar (P>0.05) stages of compaction and blastocyst development. In the first study, only the test embryos incubated with 10 cat or 20 mouse companion embryos achieved blastocyst expansion. The average total cell number within test embryos incubated with 10 cat or 20 mouse companions was greater (P<0.05) than controls or those placed with 10 mouse embryos. In the second study, test embryos in all groups achieved blastocyst expansion and had more (P<0.05) total cells per embryo than the solitary controls. Inner cell mass to trophoblast cell ratio did not differ among treatments in either study. Thus, companion mouse and cattle embryos selected for excellent quality confer a benefit to singleton cat embryos, although the number of companions necessary to grant an advantage may be species dependent. If this phenomenon can be extrapolated across species, this may be an avenue for 'common animal embryos' to improve developmental potential of embryos from rare, unrelated taxa.     

GnRH agonist Lupron (leuprolide acetate) pre-treatments prevent ovulation in response to gonadotropin stimulation in the clouded leopard (Neofelis nebulosa)

In many species, controlling the ovary prior to induction of ovulation improves the success of ovarian response and artificial insemination (AI). We assessed the impact of suppression of estrus with the GnRH agonist, Lupron, on ovarian sensitivity to equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in the clouded leopard. Seven female clouded leopards were given two injections of Lupron (3.75 mg IM) 23 d apart, followed 44 d later by eCG and hCG. Daily fecal samples were collected from 60 d before Lupron to 60 d after hCG. Fecal metabolites of estrogen (E) and progesterone (P) were measured by radioimmunoassay. Lupron decreased (P < 0.05) the number of E peaks during Lupron treatment compared to pre-Lupron. All females had baseline E and six of seven (86%) had nadir P on day of eCG. Exogenous gonadotropins induced E elevations in all females. However, mean E in the gonadotropin-provoked estrus was decreased (P < 0.05) compared to pre-Lupron estrous periods. Only one of seven (14%) females ovulated after eCG/hCG. In conclusion, estrous cycle control with Lupron resulted in predictable ovarian suppression prior to gonadotropin stimulation but altered ovarian sensitivity by an as yet unknown mechanism so that ovulation was inhibited, even when using a proven exogenous gonadotropin protocol.     

Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.     

Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival

Sperm cryopreservation is a valuable tool for the genetic management of ex situ populations. This study was conducted to assess: (1) semen characteristics of wild-born cheetahs; and (2) the impact of three types of glycerol influence (duration of exposure, temperature, and method of addition) on sperm cryosensitivity. To evaluate the impact of duration of glycerol exposure, spermatozoa were incubated in Test Yolk Buffer (TYB) with 4% glycerol at ambient temperature (approximately 22 degrees C) for 15 vs. 60 min before cryopreservation. To evaluate the influence of temperature and method of glycerol addition, spermatozoa were resuspended at ambient temperature either in TYB with 0% glycerol followed by addition of 8% glycerol (1:1 v/v; at ambient temperature vs. 5 degrees C) or directly in TYB with 4% glycerol. All samples were cryopreserved in straws over liquid nitrogen vapor and evaluated for sperm motility and acrosomal integrity after thawing. Semen samples (n = 23; n = 13 males) contained a high proportion (78%) of pleiomorphic spermatozoa. Ejaculates also contained a high proportion of acrosome-intact (86%) and motile spermatozoa (78%). Immediately after thawing, a significant proportion of spermatozoa retained intact acrosomes (range, 48-67%) and motility (range, 40-49%). After thawing, incubation in glycerol for 60 min at ambient temperature before freezing decreased (p < 0.05) sperm motility and acrosomal integrity at one time-point each (pre-centrifugation and post-centrifugation, respectively). However, method or temperature of glycerol addition had no (p > 0.05) impact on sperm cryosurvival. In summary, (1) wild-born cheetahs produce high proportions of pleiomorphic spermatozoa but with a high proportion of intact acrosomes; and (2) resuspension in 4% glycerol, followed by exposure for up to 60 min at ambient temperature, had minimal effect on sperm motility and acrosomal integrity after cryopreservation. Results indicate the feasibility of cryopreserving cheetah spermatozoa under field conditions, providing a user-friendly method to capture and store gametes to enhance genetic management.     

Nanosecond pulsed electric fields cause melanomas to self-destruct

We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.     

Thermophilic Temperature Optimum for Crenarchaeol Synthesis and Its Implication for Archaeal Evolution

The isoprenoid lipid crenarchaeol is widespread in hot springs of California and Nevada. Terrestrial and marine data together suggest a maximum relative abundance of crenarchaeol at ~40°C. This warm temperature optimum may have facilitated colonization of the ocean by (hyper)thermophilic Archaea and the major marine radiation of Crenarchaeota.     

Formation of reactive halide species by myeloperoxidase and eosinophil peroxidase

The formation of chloro- and bromohydrins from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine following incubation with myeloperoxidase or eosinophil peroxidase in the presence of hydrogen peroxide, chloride and/or bromide was analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These products were only formed below a certain pH threshold value, that increased with increasing halide concentration. Thermodynamic considerations on halide and pH dependencies of reduction potentials of all redox couples showed that the formation of a given reactive halide species in halide oxidation coupled with the reduction of compound I of heme peroxidases is only possible below a certain pH threshold that depends on halide concentration. The comparison of experimentally derived and calculated data revealed that Cl(2), Br(2), or BrCl will primarily be formed by the myeloperoxidase-H(2)O(2)-halide system. However, the eosinophil peroxidase-H(2)O(2)-halide system forms directly HOCl and HOBr.     

Optimization of humanized IgGs in glycoengineered Pichia pastoris

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.