High-concentration bovine serum albumin enhances fertilization ability of cold-stored rat sperm.

Cold transport of the cauda epididymides is a useful technique for shipping laboratory rat sperm. Cold transport of rat sperm avoids potential risks of microbiological infection, animal escape or death, and animal welfare issues. Previously, we reported that a cold-storage solution containing dimethyl sulfoxide and quercetin maintained the fertility of cold-stored rat sperm. However, cold-stored rat sperm exhibited a decreased fertilization rate after 24-h storage. To recover the fertility of cold-stored sperm, we focused on the effects of bovine serum albumin (BSA), a cholesterol acceptor that induces sperm capacitation. We sought to determine the optimal concentration of BSA in fertilization medium based on the fertility of cold-stored rat sperm. High concentrations of BSA (40 mg/ml) enhanced the fertilization rate of cold-stored rat sperm and maintained sperm fertility for 144 h. Embryos derived from cold-stored and BSA-treated sperm normally developed into pups after embryo transfer. In summary, high BSA concentrations enhanced the fertility of cold-stored rat sperm and prolonged the storage period to 144 h, thereby expanding the transportable region for genetically engineered rats.

Time elapsed between ovulation and insemination determines the quality of fertilized rat oocytes.

Genetically modified rats are valuable models in human disease research. We recently developed an improved system for rat sperm cryopreservation and in vitro fertilization (IVF) that facilitates the efficient production and preservation of genetically modified rats. In the IVF procedure performed using frozen-thawed rat sperm, the IVF schedule is fixed to ensure timely hormone administration and oocyte collection. To enhance the flexibility of the IVF schedule, possible periods of postovulated rat oocytes with normal fertility and developmental abilities should be determined. Therefore, in this study, we examined the fertilization and developmental ability of incubated oocytes 1-13 h after oocyte collection at 9:00 AM. The fertilization rate decreased 7 h after oocyte collection, and abnormally fertilized oocytes appeared 10 h after oocyte collection. The developmental rate also decreased 7 h after oocyte collection; however, live pups were obtained from oocytes 12 h after oocyte collection. In summary, ovulated rat oocytes exhibited a high developmental ability after IVF for up to 4 h after oocyte collection.

Observation of the in vitro fertilization process in living oocytes using frozen-thawed sperm in rats.

Previous studies have observed the fertilization process in rats using whole-mount preparation at different time-points after insemination. However, very few reports have described the various events during the fertilization process using an inverted microscope without whole-mount. Moreover, to the best of our knowledge, no reports have described the observation of changes in sperm motility associated with sperm penetration into oocytes. In this study, in vitro fertilization was performed using frozen-thawed sperm in various rat strains (SD, Wistar, LE, F344, and BN) and oocytes from the SD strain, and the process of sperm penetration into the oocytes and the subsequent development were observed. The sperm motility was assessed, and the correlation between the process of sperm penetration into the oocytes and sperm motility over time was examined. The motility of frozen sperm from the SD, Wistar, LE, and F344 increased at 2-3 h after thawing, at which time the sperm attached themselves to the zona pellucida. Sperm penetration into the zona pellucida occurred after 3-5 h, and pronuclei were formed in the cytoplasm of oocytes 5-9 h after insemination. The fertilities of frozen-thawed sperm from the SD, Wistar, LE, and F344 were 92.7%, 90.0%, 90.7%, and 68.7%, respectively. However, no increase in motility was observed after thawing of frozen sperm from the BN, and the fertility was only 21%. In addition, very few polyspermic oocytes were observed with use of frozen-thawed sperm of all strains. In summary, rats are suitable animals for the observation of sperm penetration into the oocytes, and we determined the timing of fertilization events in IVF using frozen-thawed rat sperm.

Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat.

Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen-thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.

Upregulation of NADPH Oxidase 2 Contributes to Renal Fibrosis in pcy Mice: An Experimental Model of Nephronophthisis.

BACKGROUND: DBA/2FG-pcy (pcy) mice harbor a homozygous Nphp3 missense mutation and develop nephronophthisis with renal interstitial fibrosis. Previous studies have shown that aberrant oxygen homeostasis contributes to the renal pathology in pcy mice, but the underlying molecular mechanism remains largely unknown. METHODS: pcy mice and a control strain, DBA/2N (DBA) mice, were used. Renal levels of 62 mRNAs involved in oxygen homeostasis were investigated by real-time PCR, and the resulting data were used for extraction of pathological pathways. On the basis of the genes found to be upregulated and pathway analysis, further studies were performed using immunoblotting, immunohistochemistry, and pharmacological intervention. RESULTS: In comparison with DBA mice, the levels of 18 mRNAs were altered by >2-fold in pcy mice. Pathway analysis extracted molecular pathways related to oxidative stress, inflammation, and cell adhesion. As the levels of mRNAs relevant to the NADPH oxidase 2 (NOX2) pathway were prominently (4 genes >5-fold) increased in pcy mice, we further analyzed the molecules related to this pathway. A time course study suggested that the pathway was gradually activated in pcy mice from at least 5 weeks of age. Immunohistochemistry study revealed that NOX2 protein was colocalized with a macrophage marker protein in the renal interstitium. Moreover, treatment of pcy mice with apocynin, an inhibitor of the NOX2 pathway, ameliorated the renal fibrosis. CONCLUSION: Our findings suggest that the activation of the NOX2 pathway, possibly mediated by macrophage infiltration, plays a pivotal role in progressive renal fibrosis in pcy mice.

Quercetin-treated rat sperm enables refrigerated transport with motility and fertility for five days.

Shipment of laboratory rats between animal facilities is frequently performed using special containers. However, the shipment of live animals is associated with potential risks of infectious diseases, escape and death during shipment and animal welfare issues. The transport of cold-stored sperm avoids such risks; however, there have been no reports on cold storage of rat sperm. We previously reported that dimethyl sulfoxide (DMSO) and quercetin maintained the motility and fertilising abilities of cold-stored mouse sperm stored for 10 days. The present study investigated the efficacy of DMSO and quercetin in the cold storage of rat sperm. Quercetin maintained motility and fertility of cold-stored rat sperm stored for 5 days. After in vitro fertilisation using cold-stored sperm, pronuclear and two-cell embryos developed normally to pups following embryo transfer. Therefore, we demonstrated that live pups could be obtained from sperm transported using the cold-storage system. We conclude that cold storage of rat sperm may provide an efficient system for transporting rat resources as an alternative to shipping live animals.

Author Correction: Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats.

Recently, genome-editing tools have come into common use in the field of rat research, and consequently, many genetically modified rat strains have been preserved and archived as frozen embryos. Although there have been many reports published on the topic of rat sperm cryopreservation, no report has yet provided satisfactory and acceptable protocols for the cryopreservation of rat sperm. In this study, we developed methods for both the cryopreservation of transgenic rat sperm and in vitro fertilisation using frozen sperm, which yielded high fertilisation rates.

A bell-shaped pattern of urinary aquaporin-2-bearing extracellular vesicle release in an experimental model of nephronophthisis.

The DBA/2-FG pcy (pcy) mouse is a model of human nephronophthisis, a recessive cystic kidney disease. Renal expression of aquaporin-2 (AQP2), a water channel protein, has been shown to be altered in pcy mice. However, the relationship between the renal expression and its release in urinary extracellular vesicles (uEV-AQP2), which account for most urinary AQP2, in pcy mice has remained largely unknown. In this study, we examined age-related alterations of this relationship in pcy mice. In comparison with control mice, pcy mice after the age of 14 weeks showed defective urinary concentration ability with an increase in urinary volume. Interestingly, the release of uEV-AQP2 increased progressively up to the age of 16 weeks, but at 21 weeks the release did not significantly differ from that in control mice (i.e., a bell-shaped pattern was evident). Similar results were obtained for uEV marker proteins, including tumor susceptibility gene 101 (TSG101) protein and apoptosis-linked gene 2-interacting protein X (Alix). Immunoblot analysis revealed that renal AQP2 expression increased progressively from 11 weeks, and immunohistochemistry showed that this increase was possibly due to an increase in the number of AQP2-positive cells. Analysis of mRNAs for seven types of AQP expressed in the kidney supported this notion. These data suggest that the level of uEV-AQP2 does not simply mirror the renal expression of AQP2 and that the altered release of uEV-AQP2 in pcy mice depends on the numbers of both renal AQP2-positive cells and EVs released into the urine.

Treatment of Alzheimer's disease with anti-homocysteic acid antibody in 3xTg-AD male mice.

Alzheimer's disease (AD) is an age-associated progressive neurodegenerative disorder with dementia, the exact pathogenic mechanisms of which remain unknown. We previously reported that homocysteic acid (HA) may be one of the pathological biomarkers in the brain with AD and that the increased levels of HA may induce the accumulation of intraneuronal amyloid-beta (Abeta) peptides. In this study, we further investigated the pathological role of HA in a mouse model of AD. Four-month-old prepathological 3xTg-AD mice exhibited higher levels of HA in the hippocampus than did age-matched nontransgenic mice, suggesting that HA accumulation may precede both Abeta and tau pathologies. We then fed 3-month-old 3xTg-AD mice with vitamin B6-deficient food for 3 weeks to increase the HA levels in the brain. Concomitantly, mice received either saline or anti-HA antibody intraventricularly via a guide cannula every 3 days during the course of the B6-deficient diet. We found that mice that received anti-HA antibody significantly resisted cognitive impairment induced by vitamin B6 deficiency and that AD-related pathological changes in their brains was attenuated compared with the saline-injected control group. A similar neuroprotective effect was observed in 12-month-old 3xTg-AD mice that received anti-HA antibody injections while receiving the regular diet. We conclude that increased brain HA triggers memory impairment and that this condition deteriorates with amyloid and leads to subsequent neurodegeneration in mouse models of AD.