Effect of D-aspartate and storage temperature on cryopreserved sperm quality and fertility in mice

Recent advances in genetic engineering have resulted in the generation of thousands of transgenic mouse lines. Assisted reproductive technologies have become essential for managing and archiving all these lines in repositories. Sperm cryopreservation appears to be the most economical way to archive mouse lines compared to embryo cryopreservation. However, there are still major challenges in using cryopreserved spermatozoa. In particular, cryodamage can compromise sperm integrity and fertility. Another challenge relates to the use of liquid nitrogen (LN2, -196°C) due to the inherent risk of injury, cost and potential difficulty in accessing this resource. The amino acid D-aspartate (D-Asp) improves in vitro fertilisation (IVF) rates with cryopreserved sperm in several mammalian species. However, how this compound exerts these effects remains poorly understood. In parallel, the use of -80°C freezers is an attractive alternative to LN2. It has already shown promising results with B6N sperm, but how this temperature affects the sperm integrity in other mouse strains has not been investigated. The results show that D-Asp administrated orally to B6N males improves sperm motility, morphology and maturation after cryopreservation in LN2. Some of these effects were recapitulated in sperm treated in vitro with D-Asp, showing that this compound can act directly on spermatozoa. D-Asp treatment was associated with a signature of oxidative stress, suggesting that this amino acid acts through reactive oxygen species (ROS) to exert its cryoprotective effects. In parallel, the response of a panel of widely used mouse strains to sperm cryopreservation at -80°C was assessed. IVF rates were similar when spermatozoa from B6N, CD-1, FVB and 129 were cryopreserved and stored in LN2 or in -80°C freezers. However, sperm from B6J and BALB/c mice showed a dramatic decrease in fertility when cryopreserved at -80°C, which was associated with a decline in viability. This shows variations in the sensitivity to cryopreservation at -80°C among mouse strains. Given the beneficial role of D-Asp on sperm stored in LN2, I have tested whether this compound could improve the quality of sperm cryopreserved at -80°C. D-Asp improved the motility, morphology and maturation of B6J and BALB/c sperm under this condition, showing that D-Asp alleviates the physiological limitations of cryopreservation at -80°C in these strains. D-Asp treatment could therefore be used in combination with long-term storage at -80°C to facilitate sperm cryopreservation and greatly improve the workflow of mouse repositories

Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice

Two conserved vocal central pattern generators broadly tuned for fast and slow rates generate species-specific vocalizations in Xenopus clawed frogs

Across phyla, males often produce species-specific vocalizations to attract females. Although understanding the neural mechanisms underlying behavior has been challenging in vertebrates, we previously identified two anatomically distinct central pattern generators (CPGs) that drive the fast and slow clicks of male Xenopus laevis, using an ex vivo preparation that produces fictive vocalizations. Here, we extended this approach to four additional species, X. amieti, X. cliivi, X. petersii, and X. tropicalis, by developing ex vivo brain preparation from which fictive vocalizations are elicited in response to a chemical or electrical stimulus. We found that even though the courtship calls are species-specific, the CPGs used to generate clicks are conserved across species. The fast CPGs, which critically rely on reciprocal connections between the parabrachial nucleus and the nucleus ambiguus, are conserved among fast-click species, and slow CPGs are shared among slow-click species. In addition, our results suggest that testosterone plays a role in organizing fast CPGs in fast-click species, but not in slow-click species. Moreover, fast CPGs are not inherited by all species but monopolized by fast-click species. The results suggest that species-specific calls of the genus Xenopus have evolved by utilizing conserved slow and/or fast CPGs inherited by each species

Replay of innate vocal patterns during night sleep in suboscines

Activation of forebrain circuitry during sleep has been variably characterized as 'pre- or replay' and has been linked to memory consolidation. The evolutionary origins of this mechanism, however, are unknown. Sleep activation of the sensorimotor pathways of learned birdsong is a particularly useful model system because the muscles controlling the vocal organ are activated, revealing syringeal activity patterns for direct comparison with those of daytime vocal activity. Here, we show that suboscine birds, which develop their species-typical songs innately without the elaborate forebrain-thalamic circuitry of the vocal learning taxa, also engage in replay during sleep. In two tyrannid species, the characteristic syringeal activation patterns of the song could also be identified during sleep. Similar to song-learning oscines, the burst structure was more variable during sleep than daytime song production. In kiskadees (Pitangus sulphuratus), a second vocalization, which is part of a multi-modal display, was also replayed during sleep along with one component of the visual display. These data show unambiguously that variable 'replay' of stereotyped vocal motor programmes is not restricted to programmes confined within forebrain circuitry. The proposed effects on vocal motor programme maintenance are, therefore, building on a pre-existing neural mechanism that predates the evolution of learned vocal motor behaviour.Fil: Döppler, Juan Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Peltier, Manon. University Of Utah. Department Of Biology; Estados UnidosFil: Amador, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Goller, Franz. University Of Utah. Department Of Biology; Estados Unidos. University of Münster; AlemaniaFil: Mindlin, Bernardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentin

An Oil-Based Adjuvant Improves Immune Responses Induced by Canine Adenovirus-Vectored Vaccine in Mice

There is a significant need for highly effective vaccines against emerging and common veterinary infectious diseases. Canine adenovirus type 2 (CAV2) vectors allow rapid development of multiple vaccines and have demonstrated their potential in animal models. In this study, we compared the immunogenicity of a non-replicating CAV2 vector encoding the rabies virus glycoprotein with and without MontanideTM ISA 201 VG, an oil-based adjuvant. All vaccinated mice rapidly achieved rabies seroconversion, which was associated with complete vaccine protection. The adjuvant increased rabies antibody titers without any significant effect on the anti-CAV2 serological responses. An RT2 Profiler™ PCR array was conducted to identify host antiviral genes modulated in the blood samples 24 h after vaccination. Functional analysis of differentially expressed genes revealed the up-regulation of the RIG-I, TLRs, NLRs, and IFNs signaling pathways. These results demonstrate that a water-in-oil-in-water adjuvant can shape the immune responses to an antigen encoded by an adenovirus, thereby enhancing the protection conferred by live recombinant vaccines. The characterization of early vaccine responses provides a better understanding of the mechanisms underlying the efficacy of CAV2-vectored vaccines

Developmental system drift in one tooth facilitates the adaptation of the other

Serial organs, such as limbs or teeth, develop with the same sets of genes and regulatory sequences. Correlated evolution is expected by default, but decoupled evolution is often achieved, as in mouse with a morphological innovation in the upper, but not the lower molar. We studied developmental evolution of hamster and mouse molars with transcriptome data. We reveal a combination of three morphogenetic changes likely causing the new morphology of the mouse upper molar. Surprisingly, most of these changes are common with the lower molar and lower molar gene expression diverged as much as, and coevolved with, the upper molar. Hence, adaptation of the upper molar has involved changes in pleiotropic genes that also modified lower molar development but preserved its final phenotype. From the lower molar point of view, it is a case of Developmental systems drift (DSD). We think that DSD accommodates pleiotropy, explaining why in teeth and more generally in the body, the evolution of developmental gene expression is fast and correlates between organs

Comparison of developmental genome expression in rodent molars reveals extensive developmental system drift

Abstract In evolution, it is widely believed that phenotypic changes root in developmental changes and phenotype conservation, in developmental conservation. Seeming phenotype conservation may however hide evolutionary changes in the underlying developmental mechanisms by which a trait is produced. This cryptic evolution is also called Developmental System Drift, and the extent of this phenomenon unclear. We used a well-characterized evo-devo model system, rodent molars, to test the correlation between phenotypic and developmental evolution. Between mouse and hamster, the morphology of the lower molars has much less diverged than the morphology of the upper molars. Is development accordingly more conserved? We compared molar crown formation with a standard approach, and with a tight transcriptome time-series to get a quantitative molecular profiling of developmental states. Our data identify common trends in the development of all molars. Upper and lower molars have their specificities since the early steps of morphogenesis, at the levels of the pattern of cusp formation, cellular composition and biased gene expression. The extent of difference in lower vs. upper molar development within one species does correlate with the extent of difference in final morphology. However, the specificity of lower vs. upper molar development is drowned among the rapid evolution of development, which is highly species-specific in term of expression levels and temporal profiles. Divergence in developmental systems is almost as high for lower as it is for upper molar, despite much lesser morphological changes in lower molar crown. Hence, our results point an extensive drift in this developmental system. Because serial organs are largely sharing gene networks, it supports previous theoretical work that suggest a causal link between pleiotropy and DSD

Oral D-Aspartate Treatment Improves Sperm Fertility in Both Young and Adult B6N Mice

Simple Summary Investigations concerning the impact of D-Aspartate on fertility suggest that it has a positive influence on the in vitro fertilization rate in young C57BL/6N mice. Here, we demonstrated that adult C57BL/6N mice that received an oral treatment of D-Aspartate also have a higher fertilizing capability and the quality of their spermatozoa increased after only two weeks of treatment. Hence, this study gives us new insights on the role of D-Aspartate in the regulation of the reproductive activity in both young and adult mice. D-Aspartate (D-Asp) treatment improved the fertility of young male C57BL/6N mice in vivo revealing a direct role on capacitation, acrosome reaction, and fertility in vitro in young males only. We investigated whether the positive effect of D-Asp on fertility could be extended to adult males and evaluated the efficacy of a 2- or 4-week-treatment in vivo. Therefore, 20 mM sodium D-Asp was supplied in drinking water to males of different ages so that they were 9 or 16 weeks old at the end of the experiments. After sperm freezing, the in vitro fertilization (IVF) rate, the birth rate, hormone levels (luteinizing hormone (LH), epitestosterone, and testosterone), the sperm quality (morphology, abnormalities, motility, and velocity), the capacitation rate, and the acrosome reaction were investigated. Oral D-Asp treatment improves the fertilizing capability in mice regardless of the age of the animals. Importantly, a short D-Asp treatment of 2 weeks in young males elevates sperm parameters to the levels of untreated adult animals. In vivo, D-Asp treatment highly improves sperm quality but not sperm concentration. Therefore, D-Asp plays a beneficial role in mouse male fertility and may be highly relevant for cryorepositories to improve mouse sperm biobanking