Despite numerous studies on mammalian fertilization, the mechanisms of fertilization—including the timing of acrosome reaction—remain largely unknown; more accurately described, the classical theory built upon years of layered experimental data is being challenged by recent conflicting evidence provided by gene-manipulated animals. Although in vitro fertilization remains our central research tool, the classical theory’s decline reminds us of the importance of in vivo observations. Here, I describe the essential roles of gene-manipulated animals in elucidating the mechanism of fertilization and the pitfalls of in vitro fertilization studies trapping many researchers.
Tupaias, or tree shrews, are small mammals that are similar in appearance to squirrels. The morphological and behavioral characteristics of the group have been extensively characterized, and despite previously being classified as primates, recent studies have placed the group in its own family, the Tupaiidae. Genomic analysis has revealed that the genus Tupaia is closer to humans than it is to rodents. In addition, tupaias are susceptible to hepatitis B virus and hepatitis C virus. The only other experimental animal that has been demonstrated to be sensitive to both of these viruses is the chimpanzee, but restrictions on animal testing have meant that experiments using chimpanzees have become almost impossible. Consequently, the development of the tupaia for use as an animal infection model could become a powerful tool for hepatitis virus research and in preclinical studies on drug development.
In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.
Exposure to organophosphate insecticides such as fenitrothion (FNT) in agriculture and public health has been reported to affect sperm quality. Antioxidants may have a potential to reduce spermatotoxic effects induced by organophosphate. The present study was carried out to evaluate the effects of palm oil tocotrienol-rich fraction (TRF) in reducing the detrimental effects occurring in spermatozoa of FNT-treated rats. Adult male Sprague-Dawley rats were divided into four equal groups: a control group and groups of rats treated orally with palm oil TRF (200 mg/kg), FNT (20 mg/kg) and palm oil TRF (200 mg/kg) combined with FNT (20 mg/kg). The sperm characteristics, DNA damage, superoxide dismutase (SOD) activity, and levels of reduced glutathione (GSH), malondialdehyde (MDA), and protein carbonyl (PC) were evaluated. Supplementation with TRF attenuated the detrimental effects of FNT by significantly increasing the sperm counts, motility, and viability and decreased the abnormal sperm morphology. The SOD activity and GSH level were significantly increased, whereas the MDA and PC levels were significantly decreased in the TRF+FNT group compared with the rats receiving FNT alone. TRF significantly decreased the DNA damage in the sperm of FNT-treated rats. A significant correlation between abnormal sperm morphology and DNA damage was found in all groups. TRF showed the potential to reduce the detrimental effects occurring in spermatozoa of FNT-treated rats.
The articular disc in the temporomandibular joint plays an important role in mandibular growth. Functional appliances induce regeneration of the condyle even after condylectomy. The aim of this study was to examine the role of the articular disc in regeneration of the condyle after unilateral condylectomy with use of a functional appliance in growing rats. Fifty growing rats were subjected to unilateral condylectomy and then half of them underwent discectomy. The functional appliance was applied to half of the rats in each group to induce regeneration of the condyle. Four and eight weeks later, morphometric and histologic analyses of the mandible were performed. Regeneration of the condyle was demonstrated in the two condylectomy groups. In the condylectomy+appliance group, the shape and cartilage of the condyle were equivalent to a normal condyle. However, regeneration of the condyle was not observed in the two discectomy groups even with the use of the functional appliance. The articular disc appears to be crucial in the regeneration of a damaged condyle, suggesting that defects or damage to the articular disc may influence mandibular growth and regeneration or repair of the condyle.
The aim of this study was to successfully establish an orthotopic murine model using two different human pancreatic adenocarcinoma cell lines and to propose a 3.0 tesla MRI protocol for noninvasive characterization of this model. SW1990 and MIAPaca-2 tumor cells were injected into the pancreas of BALB/C nu/nu mice. Tumor growth rate and morphological information were assessed by 3.0 tesla MRI (T1WI, T2WI and DCE-MRI) and immunohistology. Proliferation of SW1990 was significantly faster than that of MIAPaca-2 (P=0.000), but MIAPaca-2 mice had a significantly shorter survival than SW1990 mice (41 days and 44 days respectively, P=0.027). MRI could reliably monitor tumor growth in both cell lines: the tumors exhibiting a spherical growth pattern showed a high-intensity signal, and the SW1990 group developed significantly larger tumors compared with the MIAPaCa-2 group. There were no statistical differences between the two groups in which tumor size was assessed using electronic calipers and an MRI scan (P=0.680). Both tumors showed a slow gradual enhancement pattern. Immunohistochemistry demonstrated tumor tissues showing high expression of Ki-67. This model closely mimics human pancreatic cancer and permits monitoring of tumor growth and morphological information by noninvasive 3.0 tesla MRI studies reducing the number of mice required.
Rhubarb is often used to establish chronic diarrhea and spleen (Pi)-deficiency syndrome animal models in China. In this study, we utilized the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) method to detect changes in bacterial diversity in feces and the bowel mucosa associated with this model. Total microbial genomic DNA from the small bowel (duodenum, jejunum, and ileum), large bowel (proximal colon, distal colon, and rectum), cecum, and feces of normal and rhubarb-exposed rats were used as templates for the ERIC-PCR analysis. We found that the fecal microbial composition did not correspond to the bowel bacteria mix. More bacterial diversity was observed in the ileum of rhubarb-exposed rats (P<0.05). Furthermore, a 380 bp product was found to be increased in rhubarb-exposed rats both in faces and the bowel mucosa. The product was cloned and sequenced and showed high similarity with regions of the Bacteroides genome. AS a result of discriminant analysis with the SPSS software, the Canonical Discriminant Function Formulae for model rats was established.
Nowadays, because of increasing employment of swine for experimental studies and medical training, it is hopeful to investigate novel and effective anaesthetic protocols for preserving the animal welfare in medical investigation and concurrently improving the quality of research. Therefore, the aim of this study was to investigate a novel and effective anaesthetic protocol in swine undergoing major surgery, by translating know-how of combined anaesthesia from human protocols. Seven landrace swine were anaesthetized for three hours by a combined trial anaesthetic protocol (sedation: medetomidine, acepromazine, atropine and tramadol; induction: propofol, medetomidine and acepromazine; anaesthesia: isofluorane, propofol, medetomidine and acepromazine) and both clinical and haemodynamic parameters were compared with those of five swine anaesthetized with a control protocol (sedation: diazepam, ketamine and atropina; induction: diazepam and ketamine; anaesthesia: isofluorane). Both cardiac frequency (CF) and mean blood pressure (MBP) were significantly (P<0.05) more stable in trial protocol (CF: 78.3 ± 4.6-81.1 ± 5, MBP: 63.9 ± 10.7-96.4 ± 13.0) compared to control protocol (CF: 93.7 ± 5.5-102.5 ± 8.5, MBP: 71.0 ± 6.6-108.7 ± 7.2). The body temperature remained stable in trial protocol (°C: 36.9 ± 0.7-37.2 ± 0.3) compared to control anaesthesia (°C: 36.4 ± 0.3-37.3 ± 0.2, P<0.05). Haematosis improved undergoing combined anaesthesia (+2%, P<0.05) whereas did not change in control animals. There were no differences in respiratory rate between trial and control protocols. This study demonstrates that the proposed balanced intravenous-inhalant protocol permits to carry out a very effective, stable and safe anaesthesia in swine undergoing deep anaesthesia.
The inhalation of many types of chemicals is a leading cause of allergic respiratory diseases, and effective protocols are needed for the detection of environmental chemical–related respiratory allergies. In our previous studies, we developed a method for detecting environmental chemical–related respiratory allergens by using a long-term sensitization–challenge protocol involving BALB/c mice. In the current study, we sought to improve our model by characterizing strain-associated differences in respiratory allergic reactions to the well-known chemical respiratory allergen glutaraldehyde (GA). According to our protocol, BALB/c, NC/Nga, C3H/HeN, C57BL/6N, and CBA/J mice were sensitized dermally with GA for 3 weeks and then challenged with intratracheal or inhaled GA at 2 weeks after the last sensitization. The day after the final challenge, all mice were euthanized, and total serum IgE levels were assayed. In addition, immunocyte counts, cytokine production, and chemokine levels in the hilar lymph nodes (LNs) and bronchoalveolar lavage fluids (BALF) were also assessed. In conclusion, BALB/c and NC/Nga mice demonstrated markedly increased IgE reactions. Inflammatory cell counts in BALF were increased in the treated groups of all strains, especially BALB/c, NC/Nga, and CBA/J strains. Cytokine levels in LNs were increased in all treated groups except for C3H/HeN and were particularly high in BALB/c and NC/Nga mice. According to our results, we suggest that BALB/c and NC/Nga are highly susceptible to respiratory allergic responses and therefore are good candidates for use in our model for detecting environmental chemical respiratory allergens.
Obesity is a public health problem that increases the risk of metabolic disease, infertility, and other chronic health problems. The present study aimed to develop a new rat model for sex hormone disorder with overweight and Ca loss by intramuscular injection of exogenous leptin (LEP). Thirty female Sprague-Dawley (SD) rats (40 days old) were injected thrice intramuscularly with LEP or keyhole limpet hemocyanin immunogen. The following analyses were performed to determine the development of appetite, overweight, reproductive related-hormones, and calcium (Ca)/phosphorus (Pi) in SD rats: measurement of Lee’s index, body weight, food intake; serum Ca, Pi, and hormone tests by enzyme-linked immunosorbent analysis; histological analysis of abdominal fat; real-time polymerase chain reaction analysis of neuropeptide Y, pro-opiomelanocortin, gonadotropin-releasing hormone (Gnrh) mRNA, and gonadotropin-releasing hormone receptor (Gnrhr) mRNA expression; and western blotting analysis of enzyme phosphatidylinositol-3-kinase (PI3K). Rats injected with LEP immunogen displayed significantly increased body weight, food intake, Lee’s index, serum LEP, serum cortisol, fat deposition in the abdomen, and decreased hormones including follicle stimulating hormone, luteinizing hormone, estradiol, cholecystokinin, and Ca. Exogenous LEP administered intramuscularly also downregulate Gnrh and PI3K. In conclusion, exogenous LEP administered intramuscularly is a novel animal model for sex hormones disorder with overweight and Ca loss in SD rats. The downregulation of PI3K and Gnrh may be involved in the development of this animal model.
Because of the difficulty to exclude possible involvement of nuclear DNA mutations, it has been a controversial issue whether pathogenic mutations in mitochondrial DNA (mtDNA) and the resultant respiration defects are involved in tumor development. To address this issue, our previous study generated transmitochondrial mice (mito-mice-ND613997), which possess the nuclear and mtDNA backgrounds derived from C57BL/6J (B6) strain mice except that they carry B6 mtDNA with a G13997A mutation in the mt-Nd6 gene. Because aged mito-mice-ND613997 simultaneously showed overproduction of reactive oxygen species (ROS) in bone marrow cells and high frequency of lymphoma development, current study examined the effects of administrating a ROS scavenger on the frequency of lymphoma development. We used N-acetylcysteine (NAC) as a ROS scavenger, and showed that NAC administration prevented lymphoma development. Moreover, its administration induced longevity in mito-mice-ND613997. The gene expression profiles in bone marrow cells indicated the upregulation of the Fasl gene, which can be suppressed by NAC administration. Given that natural-killer (NK) cells mediate the apoptosis of various tumor cells via enhanced expression of genes encoding apoptotic ligands including Fasl gene, its overexpression would reflect the frequent lymphoma development in bone marrow cells. These observations suggest that continuous administration of an antioxidant would be an effective therapeutics to prevent lymphoma development enhanced by ROS overproduction.
While carnitine has been reported to have an anti-oxidative role on the ocular surface, there has been no report on the existence of a carnitine transporter (SLC22A5) in the lens. Therefore, we investigated the carnitine transport activity of canine lens epithelial cells (LEC) and determined the molecular structure of canine SLC22A5. The carnitine transport activity was 7.16 ± 0.48 pmol/mg protein/30 min. Butyrobetaine, the analogue of carnitine, reduced 30% of the activity at 50 µM. A coding sequence of canine carnitine transporter was 1694 bp long and was predicted to encode 557 amino acid polypeptides. The deduced amino acid sequence of canine carnitine transporter showed >80% similarity to that of mouse and human. Western blot analysis detected the band at 60 kDa in the membrane of lens epithelial cells. The high content of carnitine in the lens is possibly transported from aqueous humor by SLC22A5.
Xenograft models of human hematopoiesis are essential to the study of the engraftment and proliferative potential of human hematopoietic stem cells (HSCs) in vivo. Immunodeficient mice and fetal sheep are often used as xenogeneic recipients because they are immunologically naive. In this study, we transplanted human HSCs into fetal sheep and assessed the long-term engraftment of transplanted human HSCs after birth. Fourteen sheep were used in this study. In 4 fetal sheep, HSCs were transduced with homeo-box B4 (HOXB4) gene before transplantation, which promoted the expansion of HSCs. Another 4 fetal sheep were subjected to non-myeloablative conditioning with busulfan. Seven of these 8 sheep showed successful engraftment of human HSCs (1–3% of colony-forming units) as assessed after the birth of fetal sheep (5 months post-transplantation), although HOXB4-transduced HSCs showed sustained engraftment for up to 40 months. Intact HSCs were transplanted into six non-conditioned fetal sheep, and human colony-forming units were not detected in the sheep after birth. These results suggest that, as compared with mouse models, where the short lifespan of mice limits long-term follow-up of HSC engraftment, the fetal sheep model provides a unique perspective for evaluating long-term engraftment and proliferation of human HSCs.