cAMP and the Fibrous Sheath Protein CABYR (Ca2+-Binding Tyrosine-Phosphorylation-Regulated Protein) Is Required for 4D Sperm Movement.

A new life starts with successful fertilization whereby one sperm from a pool of millions fertilizes the oocyte. Sperm motility is one key factor for this selection process, which depends on a coordinated flagellar movement. The flagellar beat cycle is regulated by Ca2+ entry via CatSper, cAMP, Mg2+, ADP and ATP. This study characterizes the effects of these parameters for 4D sperm motility, especially for flagellar movement and the conserved clockwise (CW) path chirality of murine sperm. Therefore, we use detergent-extracted mouse sperm and digital holographic microscopy (DHM) to show that a balanced ratio of ATP to Mg2+ in addition with 18 µM cAMP and 1 mM ADP is necessary for controlled flagellar movement, induction of rolling along the long axis and CW path chirality. Rolling along the sperm's long axis, a proposed mechanism for sperm selection, is absent in sea urchin sperm, lacking flagellar fibrous sheath (FS) and outer-dense fibers (ODFs). In sperm lacking CABYR, a Ca2+-binding tyrosine-phosphorylation regulated protein located in the FS, the swim path chirality is preserved. We conclude that specific concentrations of ATP, ADP, cAMP and Mg2+ as well as a functional CABYR play an important role for sperm motility especially for path chirality.

Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects.

BACKGROUND/AIMS: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. METHODS: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. RESULTS: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. CONCLUSION: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.

Intraocular pressure in the smallest primate aging model: the gray mouse lemur.

OBJECTIVE: The aim of this study was to assess the practicability of common tonometers used in veterinary medicine for rapid intraocular pressure (IOP) screening, to calibrate IOP values gained by the tonometers, and to define a reference IOP value for the healthy eye in a new primate model for aging research, the gray mouse lemur. STUDIED ANIMALS AND PROCEDURES: TonoVet® and the TonoPen™ measurements were calibrated manometrically in healthy enucleated eyes of mouse lemurs euthanized for veterinary reasons. For comparison of the practicability of both tonometers as a rapid IOP assessment tool for living mouse lemurs, the IOP of 24 eyes of 12 animals held in the hand was measured. To define a standard reference value for IOP in mouse lemurs, 258 healthy animals were measured using the TonoVet® . RESULTS: Intraocular pressure measurements for the TonoVet® can be corrected using the formula: y = 0.981 + (1.962*TonoVet® value), and those for the TonoPen™ using that of y = 5.38 + (1.426*TonoPen™ value). The calibrated IOP for a healthy mouse lemur eye was 20.3 ± 2.8 mmHg. The TonoVet® showed advantages in practicability, for example, small corneal contact area, short and painless corneal contact, shortened total time spent on investigation, as well as the more accurate measured values. IOP measurements of healthy mouse lemur eyes were not affected by age, sex, eye side, or colony. CONCLUSION: Tonometry using TonoVet® is the more practicable assessment tool for IOP measurement of the tiny eyes of living mouse lemurs. Pathological deviations can be identified based on the described reference value.

Wolfram-silicone implants as effective radiation shielding for ocular brachytherapy: dosimetric features and in vivo animal study on biocompatibility.

OBJECTIVE: To evaluate wolfram as a photon and beta absorber in the management of uveal melanoma with radiotherapy, examining its potential ocular adverse effects and physiologic tolerance using an in vivo rabbit ocular model. METHODS: A method of manufacturing implants from mixtures of wolfram and silicone was developed. Their shielding effect on the radiation of sources used in ocular brachytherapy was investigated by dosimetric measurement in an eye phantom as well as numerical simulations. Different wolfram implantation techniques, such as extraocular fixation of a wolfram-silicone implant (n = 1), vitrectomy with silicone oil and intravitreal injection of a wolfram-silicone oil suspension (n = 2), and concurrent attachment of a wolfram implant onto the sclera (n = 2), were tested to investigate the long-term effects of wolfram. A vitrectomy with silicone oil without wolfram implantation was carried out in 2 rabbits (n = 2), constituting the control group. The eyes were enucleated after 3 months for histologic analysis. RESULTS: Wolfram-silicone mixtures have been dosimetrically proven to be very effective radiation absorbers for use in ocular brachytherapy. Severe complications, such as endophthalmitis, secondary glaucoma, cornea decompensation, and vessel occlusion, were not documented in the tested rabbit eyes after the application of wolfram. Histologic examination of the bulbi after enucleation showed epiretinal gliosis without further pathologic findings in all eyes after vitrectomy. CONCLUSIONS: The results of this study show that wolfram and wolfram-silicone implants constitute a promising candidate as potential radiation shielding substrates.

Effects of castration and sterilization on baseline and response levels of cortisol-A case study in male guinea pigs.

An uncontrolled reproduction of animals in human hands should be avoided. To meet this goal, animals are widely castrated, i.e., the gonads are completely removed. Since the gonads are the most important source of sex hormones, this is a serious intervention in the entire endocrine system of an organism. Sterilization is a much less invasive procedure. Thus, it could have advantages over castration. Therefore, the overall aim of this study was to analyze the effect of castration vs. sterilization on the release of glucocorticoids, i.e., an important indicator for welfare. Taking domestic guinea pigs as a model system, we studied baseline and response cortisol values (cortisol is the main glucocorticoid in guinea pigs) in castrated, sterilized, sham-operated and intact males and baseline values in their cohoused females. Whereas baseline values of males did not differ between the groups, castrated males showed significantly higher cortisol response levels than intact, sham-operated and sterilized males. Females housed with castrated, sterilized, sham-operated or intact males did not differ in their cortisol concentrations, neither shortly after being placed with the respective male or after being co-housed for several weeks. Overall, the results support the hypothesis that castrated males exhibited a higher cortisol responsiveness during acute challenge which could point to a generalized impaired welfare of castrated males in comparison to intact, sham-operated and sterilized males. Our results provide first evidence for a potential negative impact of castration on the animals' welfare, while at the same time pointing toward sterilization representing a less invasive, promising alternative. Therefore, the results may stimulate future research on this topic to further detect potential welfare-related side effects of castration.

Spontaneous Spongiform Brainstem Degeneration in a Young Mouse Lemur (Microcebus murinus) with Conspicuous Behavioral, Motor, Growth, and Ocular Pathologies.

Here we report a case of severe growth retardation and neurologic abnormalities in a female gray mouse lemur (Microcebus murinus), a small NHP species for which the genomic sequence recently became available. The female lemur we present here died on postnatal day 125. This lemur had impaired development of motor skills and showed severe ataxia and tremors. In addition, hearing seemed normal whereas ophthalmic examination revealed incipient bilateral cataracts, abnormal pigmentation in the lens of the left eye, and a missing optokinetic nystagmus, which indicated impaired vision. Most prominently, the lemur showed severe growth retardation. Necropsy revealed maldevelopment of the left reproductive organs and unilateral dilation of the right lateral ventricle, which was confirmed on brain MRI. Brain histology further revealed large, bilateral areas of vacuolation within the brainstem, but immunohistochemistry indicated no sign of pathologic prion protein deposition. Full genomic sequencing of the lemur revealed a probably pathologic mutation in LARGE2 of the LARGE gene family, which has been associated with congenital muscular dystrophies. However, potentially functional mutations in other genes were also present. The observed behavioral and motor signs in the presented animal might have been linked to spongiform degeneration and resulting brainstem dysfunction and progressive muscle weakness. The macroscopic developmental abnormalities and ophthalmic findings might be genetic in origin and linked to the mutation in LARGE2.

A review on ocular findings in mouse lemurs: potential links to age and genetic background.

Mouse lemurs, the world's smallest primates, inhabit forests in Madagascar. They are nocturnal, arboreal and dependent on vision for their everyday lives. In the last decades, the grey mouse lemur became increasingly important for biomedical research, in particular aging research. Experiments which require the combination of visual fitness and old age consequently depend on a solid knowledge of ocular pathologies. Although ocular diseases in mouse lemurs have been described as being common, they have not received much attention so far. Yet it is important to know when and why ocular diseases in captive mouse lemurs may occur. This review aims to provide a comprehensive overview of known ocular findings in mouse lemurs. It summarizes the frequency of ocular findings in captive mouse lemur colonies and points to their likely causes and treatment options based on the evidence available from other animals and humans. In addition, it shall be discussed whether age or genetic background may affect their development. This review may be used as a reference for future studies which require an assessment of visual performance in mouse lemurs and help to evaluate observed clinical signs and ocular diseases. Furthermore, the high incidence of specific diseases may provide new perspectives and set the groundwork for a new animal model for ocular research.

Photoperiodic regime influences onset of lens opacities in a non-human primate.

BACKGROUND: Opacities of the lens are typical age-related phenomena which have a high influence on photoreception and consequently circadian rhythm. In mouse lemurs, a small bodied non-human primate, a high incidence (more than 50% when >seven years) of cataracts has been previously described during aging. Previous studies showed that photoperiodically induced accelerated annual rhythms alter some of mouse lemurs' life history traits. Whether a modification of photoperiod also affects the onset of age dependent lens opacities has not been investigated so far. The aim of this study was therefore to characterise the type of opacity and the mouse lemurs' age at its onset in two colonies with different photoperiodic regimen. METHODS: Two of the largest mouse lemur colonies in Europe were investigated: Colony 1 having a natural annual photoperiodic regime and Colony 2 with an induced accelerated annual cycle. A slit-lamp was used to determine opacities in the lens. Furthermore, a subset of all animals which showed no opacities in the lens nucleus in the first examination but developed first changes in the following examination were further examined to estimate the age at onset of opacities. In total, 387 animals were examined and 57 represented the subset for age at onset estimation. RESULTS: The first and most commonly observable opacity in the lens was nuclear sclerosis. Mouse lemurs from Colony 1 showed a delayed onset of nuclear sclerosis compared to mouse lemurs from Colony 2 (4.35 ± 1.50 years vs. 2.75 ± 0.99 years). For colony 1, the chronological age was equivalent to the number of seasonal cycles experienced by the mouse lemurs. For colony 2, in which seasonal cycles were accelerated by a factor of 1.5, mouse lemurs had experienced 4.13 ± 1.50 seasonal cycles in 2.75 ± 0.99 chronological years. DISCUSSION: Our study showed clear differences in age at the onset of nuclear sclerosis formation between lemurs kept under different photoperiodic regimes. Instead of measuring the chronological age, the number of seasonal cycles (N = four) experienced by a mouse lemur can be used to estimate the risk of beginning nuclear sclerosis formation. Ophthalmological examinations should be taken into account when animals older than 5-6 seasonal cycles are used for experiments in which unrestricted visual ability has to be ensured. This study is the first to assess and demonstrate the influence of annual photoperiod regime on the incidence of lens opacities in a non-human primate.

Spontaneous listeriosis in grey mouse lemurs (Microcebus murinus), but not in Goodman's mouse lemurs (Microcebus lehilahytsara) of the same colony.

Listeriosis is a zoonotic infection with the gram positive, facultative intracellular bacterium Listeria (L.) monocytogenes. Infections mainly occur in ruminants, but also in other species, including humans. Case fatality rate usually is high. The incidence of listeriosis in captive non-human primates is very low. We report the first spontaneous, fatal, and likely food-born outbreak of listeriosis in a population of captive grey mouse lemurs (Microcebus murinus). Conspicuously, none of the closely related Goodman's mouse lemurs (Microcebus lehilahytsara) in the same facility were affected.