Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F 1 hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX MSM , which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX MSM males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX MSM background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX MSM spermatozoa have a defect in penetration through the zona pellucida of eggs.

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.

Mutations of the leucine-rich glioma-inactivated 1 (LGI1) gene cause an autosomal dominant partial epilepsy with auditory features also known as autosomal-dominant lateral temporal lobe epilepsy. LGI1 is also the main antigen present in sera and cerebrospinal fluids of patients with limbic encephalitis and seizures, highlighting its importance in a spectrum of epileptic disorders. LGI1 encodes a neuronal secreted protein, whose brain function is still poorly understood. Here, we generated, by ENU (N-ethyl-N-nitrosourea) mutagenesis, Lgi1-mutant rats carrying a missense mutation (L385R). We found that the L385R mutation prevents the secretion of Lgi1 protein by COS7 transfected cells. However, the L385R-Lgi1 protein was found at low levels in the brains and cultured neurons of Lgi1-mutant rats, suggesting that mutant protein may be destabilized in vivo. Studies on the behavioral phenotype and intracranial electroencephalographic signals from Lgi1-mutant rats recalled several features of the human genetic disorder. We show that homozygous Lgi1-mutant rats (Lgi1(L385R/L385R)) generated early-onset spontaneous epileptic seizures from P10 and died prematurely. Heterozygous Lgi1-mutant rats (Lgi1(+/L385R)) were more susceptible to sound-induced, generalized tonic-clonic seizures than control rats. Audiogenic seizures were suppressed by antiepileptic drugs such as carbamazepine, phenytoin and levetiracetam, which are commonly used to treat partial seizures, but not by the prototypic absence seizure drug, ethosuximide. Our findings provide the first rat model with a missense mutation in Lgi1 gene, an original model complementary to knockout mice. This study revealed that LGI1 disease-causing missense mutations might cause a depletion of the protein in neurons, and not only a failure of Lgi1 secretion.

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In vitro fertilization (IVF) is a valuable technique for the propagation of experimental animals. IVF has typically been used in mice to rapidly expand breeding colonies and create large numbers of embryos. However, applications of IVF in rat breeding experiments have stalled due to the inconvenient laboratory work schedules imposed by current IVF protocols for this species. Here, we developed a new rat IVF protocol that consists of experimental steps performed during common laboratory working hours. Our protocol can be completed within 12 h by shortening the period of sperm capacitation from 5 to 1 h and the fertilization time from 10 to 8 h in human tubal fluid (HTF) medium. This new protocol generated an excellent birth rate and was applicable not only to closed colony rat strains, such as Wistar, Long-Evans, and Sprague-Dawley (SD), but also to the inbred Lewis strain. Moreover, Wistar and Long-Evans embryos prepared by this protocol were successfully frozen by vitrification and later successfully thawed and resuscitated. This protocol is practical and can be easily adopted by laboratory workers.