Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a “9+2” structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes. Molecular components of the flagellar axoneme are evolutionally conserved from unicellular flagellates to mammals, including mice. Although knockout (KO) mice have been generated to understand their function in the formation and motility regulation of sperm flagella, the majority of KO mice die before sexual maturation due to impaired ciliary motility, which makes it challenging to analyze mature spermatozoa. In this review, we introduce methods that have been used to overcome premature lethality, focusing on KO mouse lines of central pair components.

Our early weaning schedule was associated with the emergence of trait anxiety in male rodents performing an elevated plus maze but not an open-field test. We previously reported that early weaning weakened excitatory neurotransmission to the amygdala from the prefrontal cortex, where the mesocorticolimbic dopaminergic (DAergic) fiber terminates on each. In this study, we investigated DAergic transmission in both these brain regions. The extracellular levels of amygdalar DA in adulthood were two times higher in rats weaned at 16 days compared to those weaned at 30 days in both the home cage and the open-field. This difference in extracellular DA levels was not apparent in the prefrontal cortex. The concurrently measured locomotor and rearing behaviors did not vary according to the weaning period and the probe-implanted region, respectively. These results suggest that the effects of early weaning on DA tone appear to be specific to the amygdala and do not represent ubiquitous upregulation as these changes were not observed in the prefrontal cortex.

We recently demonstrated that aspartoacylase (Aspa) and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) genes were causative of essential tremor (ET) in rats. This finding was obtained using Aspa^em34Kyo/Hcn1^A354V double-mutant rats, but they were bred on a heterogeneous genetic background of two strains, F344 and WTC. Here, we developed an Aspa^em34Kyo/Hcn1^em1Kyo double-knockout rat strain with a homogenous F344 genetic background and studied the ability of glutamate receptor antagonists to suppress ET. The F344-Aspa/Hcn1 double-knockout rats exhibited spontaneous, intense body tremor equivalent to that in the double-mutant rats. N-acetyl-aspartate (NAA), a substrate of ASPA, showed accumulation in all brain regions and in the spinal cord. However, N-acetyl-aspartyl-glutamate (NAAG), which is derived from NAA and interacts with glutamatergic receptors, was decreased in the medulla oblongata of the double-knockout rats. The tremor was suppressed by 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, in F344-Aspa/Hcn1 double-knockout rats. The non-NMDA glutamate receptor antagonist NBQX weakly inhibited the tremor, while the metabotropic glutamate receptor antagonist LY341495 showed no effect. In addition, both NR2B subunit-specific (Ro 25-6981) and NR2C/NR2D subunit-specific (cis-piperidine dicarboxylic acid) NMDA receptor antagonists suppressed the tremor. These data indicated that the pathogenesis of tremor in Aspa/Hcn1 double-knockout rats involved ionotropic glutamate receptors, particularly NMDA receptors.

Gender and menopause influence the severity and development manner of nonalcoholic steatohepatitis (NASH). Male p62/Sqstm1 and nuclear factor E2-related factor-2 (p62 and Nrf2) double-knockout (DKO) mice exhibit severe steatohepatitis caused by hyperphagia-induced obesity, overload of lipopolysaccharide (LPS) into the liver, and potentiation of the inflammatory response in Kupffer cells. However, the pathogenetic phenotype of steatohepatitis in female DKO mice remains unknown. Phenotypic changes of steatohepatitis in DKO mice were compared in terms of gender differences. Compared with DKO male mice, DKO female mice exhibited later onset of steatohepatitis with obesity after 30 weeks of age, as well as milder severity of hepatic inflammation and fibrosis. Serum estradiol was higher in female than male mice, with levels increasing up to 30 weeks of age before decreasing until 50 weeks of age (corresponding to the post-menopausal period). Fecal and serum LPS were lower in female mice than male mice, and inflammatory signaling in the liver was attenuated in female compared with male mice. Correlating with LPS levels, the composition of intestinal microbiota in female mice was different from male mice. Gender differences were observed for the development of steatohepatitis in DKO mice. Low-grade inflammatory hit in the liver under in vivo conditions of high estradiol may be attributable to the milder pathological features of steatohepatitis in female mice.

Ehlers–Danlos syndromes (EDSs) are heterogeneous group of heritable connective tissue disorders characterized by joint and skin hyperextensibility as well as fragility of various organs. Recently, we described a new type of EDS, musculocontractual EDS (mcEDS-CHST14), caused by pathogenic variants of the carbohydrate sulfotransferase 14 (CHST14) gene mutation. B6;129S5-Chst14^tm1Lex/Mmucd (B6;129-Chst14 KO) mice are expected to be an animal model of mcEDS-CHST14. However, >90% of B6;129-Chst14 KO homozygous (B6;129-Chst14^−/−) mice show perinatal lethality. Therefore, improvement of the birth rate of Chst14^−/− mice is needed to clarify the pathophysiology of mcEDS-CHST14 using this animal model. Some B6;129-Chst14^−/− embryos had survived at embryonic day 18.5 in utero, suggesting that problems with delivery and/or childcare may cause perinatal lethality. However, in vitro fertilization and egg transfer did not improve the birth rate of the mice. A recent report showed that backcrossing to C57BL/6 strain induces perinatal death of all Chst14^−/− mice, suggesting that genetic background influences the birthrate of these mice. In the present study, we performed backcrossing of B6;129-Chst14 KO mice to a BALB/c strain, an inbred strain that shows lower risks of litter loss than C57BL/6 strain. Upon backcrossing 1 to 12 times, the birth rate of Chst14^−/− mice was improved with a birth rate of 6.12–18.64%. These results suggest that the genetic background influences the birth rate of Chst14^−/− mice. BALB/c congenic Chst14^−/− (BALB.Chst14^−/−) mice may facilitate investigation of mcEDS-CHST14. Furthermore, backcrossing to an appropriate strain may contribute to optimizing animal experiments.

Colorectal cancer is the second most lethal cancer type across all ages and sexes, the many mechanisms of which are still currently being further elucidated. PIERCE1 has been known to be involved in the cell cycle and proliferation, the expression of which is regulated by stress conditions in a p53-dependent manner. Through a database search, we found that PIERCE1 was significantly augmented in patients with colorectal carcinoma compared to normal samples, suggesting its possible role in tumor regulation. Recently, PIERCE1 has also been reported to increase proliferation of a liver cancer cell line, indicating its possible role as an oncogene. To examine its relevance to tumorigenesis, such as whether it has either oncogenic or tumor suppressive function, PIERCE1 was knocked down and overexpressed in several colorectal cancer cell lines and mice, respectively. To evaluate the roles of Pierce1 in vivo, we established a Pierce1 transgenic (TG) mouse model and then administered azoxymethane with dextran sodium sulfate (DSS) to induce colorectal carcinogenesis via promoting mutations in Apc and Kras. Nonetheless, PIERCE1 depletion in these cell lines showed no significant change in cell growth. AOM/DSS-treated Pierce1 TG mice were comparable with respect to colon lengths, the number of polyps, and tumor sizes to those of the control mice. These results implicate that PIERCE1 does not play an oncogenic or tumor suppressive role in AOM/DSS-induced colorectal cancer.

The cold storage of two-cell embryos is a useful technique for transporting genetically engineered mice without the shipment of live animals. However, the developmental ability of cold-stored embryos decreases with prolonged storage periods. Therefore, the transported embryos must be readily transferred to recipient mice upon arrival. The cryopreservation of cold-transported embryos may improve the flexibility of the schedule of embryo transfer. In this paper, we examined the viability and developmental ability of vitrified-warmed mouse embryos at the two-cell stage after cold storage in refrigerated temperatures for 0, 24, 48, 72, or 96 h. The viability of vitrified-warmed embryos after cold storage was comparable to vitrified-warmed embryos without cold storage. Vitrified-warmed embryos after cold storage also developed normally to pups by embryo transfer. In addition, live pups were obtained from vitrified-warmed embryos after cold-transportation from Asahikawa Medical University. In summary, cold-stored embryos can be used for the transportation and archive of genetically engineered mice.

Recent studies in mice suggested that KLF5 (Kruppel like factor 5), a zinc-finger transcription factor, plays an important role in skeletal muscle development and regeneration. As an important factor in the process of muscle development, KLF5 participates in the regulation of the cell cycle, cell survival, and cell dryness under different environmental conditions, but it is not clear whether KLF5 participates in muscle atrophy. Therefore, we investigated whether KLF5 can regulate the atrophy of chicken satellite cells in vitro and examined its mechanism of action. qPCR showed that KLF5 gene knockdown promoted the expression of key genes in muscle atrophy. Subsequently, we sequenced and analyzed the transcriptomes of KLF5 silenced and control cells, and we showed that the differentially expressed genes were mainly enriched in 10 signaling pathways (P<0.05), with differential gene and enrichment analyses indicating that the Wnt signaling pathways are extremely important. In conclusion, our results indicate that KLF5 may regulate the atrophy of chicken skeletal muscle through the Wnt/β-catenin signaling pathway.

Among the intact male rats, a subpopulation has been found to show little or no sexual behavior, even after experiencing several mating sessions. This study investigated whether sexually sluggish (SS) males show behavioral differences from normal copulatory (NC) males, other than those concerning sexual behavior. The olfactory preference of males was measured through the time spent displaying nose-poking behavior directed at sexually active males and estrous females for odor exploration in a three-chamber apparatus. Both the NC and SS males showed a significant preference for the odor of estrous females compared with that of male odors. However, SS males spent significantly less time nose-poking estrous females than NC males. The food-finding test was performed after overnight fasting. Our findings showed that all the NC males found the buried pellet within 5 min, whereas over 60% of the SS males failed to find it. The males were also tested for their ability to find a buried bag containing soiled bedding from estrous female cages. The bag was found by 80% of NC males, but only by 20% of SS males. Our results suggest that SS and NC male rats differ not only in sexual behavior but also in other functions such as olfaction.

The non-motor symptoms (NMS) of Parkinson’s disease (PD) are found in more than 90% of patients with PD. Here, we explored the effects of electroacupuncture (EA) stimulation at Zhong wan (CV-12), Qihai (RN-7), Zusanli (ST-36) and Taichong (LR-3) on NMS and brain-gut peptides of PD. We found that EA intervention alleviated the motor deficit induced by 6-OHDA in rats indicated by the decreased abnormal involuntary movements (AIMs) scores and the net number of rotations and increased cylinder test grade. It also improved the spatial memory and attenuated anxiety-like and depression of PD model rats. EA treatment significantly inhibited neuronal apoptosis in PD model animals, as demonstrated by the increased number of TH positive cells and reduced number of apoptotic cells in the substantia nigra. The expression of cleaved caspase-3 and cleaved PARP in PD model rats was markedly suppressed by EA stimulation. Moreover, EA remarkably inhibited the inflammatory response in PD model rats, as revealed by the decreased levels of TNF-α, IL-1β, and COX-2 mRNA expression. It also attenuated the oxidative stress in rats, as indicated by the increased levels of SOD and GSH and the decreased level of MDA. EA treatment contributed to alleviating PD by regulating brain-gut peptides in rats, such as NPY, CCK, SST, GAS, and PYY. In conclusion, EA stimulation at CV-12, RN-7, ST-36, and LR-3 effectively alleviates the NMS of PD partly through regulating the levels of brain-gut peptides.