Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus.

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals.

Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

BACKGROUND AND OBJECTIVE: Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. METHODS: Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. RESULTS: After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. CONCLUSIONS: Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain.

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95 %. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6 %, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.

Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus.

Motilin and ghrelin are the gastrointestinal (GI) hormones released in a fasting state to stimulate the GI motility of the migrating motor complex (MMC). We focused on coordination of the ghrelin/motilin family in gastric contraction in vivo and in vitro using the house musk shrew (Suncus murinus), a ghrelin- and motilin-producing mammal. To measure the contractile activity of the stomach in vivo, we recorded GI contractions either in the free-moving conscious or anesthetized S. murinus and examined the effects of administration of motilin and/or ghrelin on spontaneous MMC in the fasting state. In the in vitro study, we also studied the coordinative effect of these hormones on the isolated stomach using an organ bath. In the fasting state, phase I, II, and III contractions were clearly recorded in the gastric body (as observed in humans and dogs). Intravenous infusion of ghrelin stimulated gastric contraction in the latter half of phase I and in the phase II in a dose-dependent manner. Continuous intravenous infusion of ghrelin antagonist (d-Lys3-GHRP6) significantly suppressed spontaneous phase II contractions and prolonged the time of occurrence of the peak of phase III contractions. However, intravenous infusion of motilin antagonist (MA-2029) did not inhibit phase II contractions but delayed the occurrence of phase III contractions of the MMC. In the in vitro study, even though a high dose of ghrelin did not stimulate contraction of stomach preparations, ghrelin administration (10(-10)-10(-7) M) with pretreatment of a low dose of motilin (10(-10) M) induced gastric contraction in a dose-dependent manner. Pretreatment with 10(-8) M ghrelin enhanced motilin-stimulated gastric contractions by 10 times. The interrelation of these peptides was also demonstrated in the anesthetized S. murinus. The results suggest that ghrelin is important for the phase II contraction and that coordination of motilin and ghrelin are necessary to initiate phase III contraction of the MMC.

Whole-genome resequencing shows numerous genes with nonsynonymous SNPs in the Japanese native cattle Kuchinoshima-Ushi.

BACKGROUND: Because the Japanese native cattle Kuchinoshima-Ushi have been isolated in a small island and their lineage has been intensely protected, it has been assumed to date that numerous and valuable genomic variations are conserved in this cattle breed. RESULTS: In this study, we evaluated genetic features of this breed, including single nucleotide polymorphism (SNP) information, by whole-genome sequencing using a Genome Analyzer II. A total of 64.2 Gb of sequence was generated, of which 86% of the obtained reads were successfully mapped to the reference sequence (Btau 4.0) with BWA. On an average, 93% of the genome was covered by the reads and the number of mapped reads corresponded to 15.8-fold coverage across the covered region. From these data, we identified 6.3 million SNPs, of which more than 5.5 million (87%) were found to be new. Out of the SNPs annotated in the bovine sequence assembly, 20,432 were found in protein-coding regions containing 11,713 nonsynonymous SNPs in 4,643 genes. Furthermore, phylogenetic analysis using sequence data from 10 genes (more than 10 kbp) showed that Kuchinoshima-Ushi is clearly distinct from European domestic breeds of cattle. CONCLUSIONS: These results provide a framework for further genetic studies in the Kuchinoshima-Ushi population and research on functions of SNP-containing genes, which would aid in understanding the molecular basis underlying phenotypic variation of economically important traits in cattle and in improving intrinsic defects in domestic cattle breeds.

Immunolocalization of spetex-1 at the connecting piece in spermatozoa of the musk shrew (Suncus murinus).

Spetex-1, which has been isolated by differential display and rat cDNA library screening as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs that locates at both the segmented column in the connecting piece and outer dense fibers-affiliated satellite fibrils in rat sperm flagella. Orthologs of Spetex-1 are identified in many animal species, including human, chimpanzee, macaque, cow, dog, African clawed frog, green spotted puffer, and zebrafish. In this study, we used RT-PCR in combination with 5' and 3' RACE (Rapid Amplification of cDNA End) technique to isolate Spetex-1 ortholog of the musk shrew (Suneus murinus), which yielded a full-length Suncus Spetex-1 gene containing an open reading frame of 1,908 base pairs encoding a protein of 636 amino acids with the predicted molecular mass of 72,348 Da. Suncus Spetex-1 has two coiled-coil motifs at 118-184 and 242-276 amino acid residues, which is a characteristic shared by mammalian Spetex-1 proteins. To examine the subcellular localization of Spetex-1 in Suncus spermatozoa, we produced the anti-Suncus Spetex-1 antibody and carried out immunocytochemistry. In spite of that the primary structure of Suncus Spetex-1 is basically similar to that of rat and mouse Spetex-1, confocal laser scanning microscopy and immunoelectron microscopy revealed that Spetex-1 was restricted to the segmented column and capitulum in the connecting piece of Suncus spermatozoa and was not detected in other parts of flagella, suggesting a diversity of Spetex-1 localization in mammalian spermatozoa.

Lineage-specific duplication and loss of pepsinogen genes in hominoid evolution.

Fourteen different pepsinogen-A cDNAs and one pepsinogen-C cDNA have been cloned from gastric mucosa of the orangutan, Pongo pygmaeus. Encoded pepsinogens A were classified into two groups, i.e., types A1 and A2, which are different in acidic character. The occurrence of 9 and 5 alleles of A1 and A2 genes (at least 5 and 3 loci), respectively was anticipated. Respective orthologous genes are present in the chimpanzee genome although their copy numbers are much smaller than those of the orangutan genes. Only A1 genes are present in the human probably due to the loss of the A2 gene. Molecular phylogenetic analyses showed that A1 and A2 genes diverged before the speciation of great hominoids. Further reduplications of respective genes occurred several times in the orangutan lineage, with much higher frequencies than those occurred in the chimpanzee and human lineages. The rates of non-synonymous substitutions were higher than those of synonymous ones in the lineage of A2 genes, implying the contribution of the positive selection on the encoded enzymes. Several sites of pepsin moieties were indeed found to be under positive selection, and most of them locate on the surface of the molecule, being involved in the conformational flexibility. Deduced from the known genomic structures of pepsinogen-A genes of primates and other mammals, the duplication/loss were frequent during their evolution. The extreme multiplication in the orangutan might be advantageous for digestion of herbaceous foods due to the increase in the level of enzymes in stomach and the diversification of enzyme specificity.

Physiological characteristics of gastric contractions and circadian gastric motility in the free-moving conscious house musk shrew (Suncus murinus).

Although many studies have demonstrated the physiological action of motilin on the migrating motor complex, the precise mechanisms remain obscure. To obtain new insights into the mechanisms, we focused on the house musk shrew (Suncus murinus, suncus used as a laboratory name) as a small model animal for in vivo motilin study, and we studied the physiological characteristics of suncus gastrointestinal motility. Strain gauge transducers were implanted on the serosa of the gastric body and duodenum, and we recorded gastrointestinal contractions in the free-moving conscious suncus and also examined the effects of intravenous infusion of various agents on gastrointestinal motility. During the fasted state, the suncus stomach and duodenum showed clear migrating phase III contractions (intervals of 80-150 min) as found in humans and dogs. Motilin (bolus injection, 100-300 ng/kg; continuous infusion, 10-100 ng·kg(-1)·min(-1)) and erythromycin (80 µg·kg(-1)·min(-1)) induced gastric phase III contractions, and motilin injection also increased the gastric motility index in a dose-dependent manner (P < 0.05, vs. saline). Pretreatment with atropine completely abolished the motilin-induced gastric phase III contractions. On the other hand, in the free-feeding condition, the suncus showed a relatively long fasting period in the light phase followed by spontaneous gastric phase III contractions. The results suggest that the suncus has almost the same gastrointestinal motility and motilin response as those found in humans and dogs, and we propose the suncus as a new small model animal for studying gastrointestinal motility and motilin in vivo.

House musk shrew (Suncus murinus, order: Insectivora) as a new model animal for motilin study.

Although many studies have demonstrated the action of motilin on migrating motor complex by using human subjects and relatively large animals, the precise physiological mechanisms of motilin remain obscure. One reason for the lack of progress in this research field is that large animals are generally not suitable for molecular-level study. To overcome this problem, in this study, we focused on the house musk shrew (Suncus murinus, order: Insectivora, suncus named as laboratory strain) as a small model animal, and we present here the results of motilin gene cloning and its availability for motilin study. The motilin gene has a high homology sequence with that of other mammals, including humans. Suncus motilin is predicted to exist as a 117-residue prepropeptide that undergoes proteolytic cleavage to form a 22-amino-acid mature peptide. The results of RT-PCR showed that motilin mRNA is highly expressed in the upper small intestine, and low levels of expression were found in many tissues. Morphological analysis revealed that suncus motilin-producing cells were present in the upper small intestinal mucosal layer but not in the myenteric plexus. Administration of suncus motilin to prepared muscle strips of rabbit duodenum showed almost the same contractile effect as that of human motilin. Moreover, suncus stomach preparations clearly responded to suncus or human motilin stimulation. To our knowledge, this is the first report that physiological active motilin was determined in small laboratory animals, and the results of this study suggest that suncus is a suitable model animal for studying the motilin-ghrelin family.

Sensitivity of liver injury in heterozygous Sod2 knockout mice treated with troglitazone or acetaminophen.

Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2+/-) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2+/- mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2+/- mice. Although we conducted a repeated dose toxicity study in Sod2+/- mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/- mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2+/- mice. These results suggest that Sod2+/- mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.

Scanning transmission electron microscopic analysis of nitrogen generated by 3, 3'-diaminobenzidine-besed peroxidase reaction with resin ultrathin sections of rhinoceros parotid gland acinar cells.

A 3, 3'-diaminobenzidine (DAB)-based method was used to detect the localization of endogenous peroxidase activity in Indian rhinoceros (Rhinoceros unicornis) parotid gland acinar cells. The tissue had previously been resin-embedded in gelatin capsules for routine electron microscopic observations and thus pre-incubation for endogenous peroxidase analysis was not possible. We attempted to demonstrate the relationship between secretory granules (SGs) in resin ultrathin sections of Indian rhinoceros parotid gland acinar cells and endogenous peroxidase activity. A JEM 1400 Plus scanning transmission electron microscope (STEM) was used to conduct energy dispersive X-ray spectroscopy (EDS) analysis of the presence of nitrogen generated by the DAB reaction in bipartite structural SG consisting of a dense body (or core). The mapping patterns of nitrogen were restricted to the dense body. We observed nitrogen localized in the rough endoplasmic reticulum (ER), nuclear envelope (NE) and several components of the Golgi apparatus (G) of rhinoceros parotid gland acinar cells participating in the synthetic pathway of secretory proteins. Moreover, we established a nitrogen-detection method by EDS analysis of rhinoceros parotid gland. The reliability of the method was validated by comparison of the test group (peroxidase detection in ultrathin resin sections) and the control group (ordinary peroxidase detection in semi-thin sections following glutaraldehyde pre-fixation) of rat submandibular gland. The same mapping patterns of nitrogen were detected by DAB reaction in the SG, ER, NE and G in these two groups. Hence, EDS-STEM approaches for endogenous peroxidase post-incubation analysis will prove useful for advanced cytochemical analysis for the identification of any other resin sections.

Insertion of an intracisternal A particle retrotransposon element in plasma membrane calcium ATPase 2 gene attenuates its expression and produces an ataxic phenotype in joggle mutant mice.

Using forward genetic analysis, we identified the insertion of an intracisternal A particle (IAP) retrotransposon element in the plasma membrane calcium ATPase 2 gene (Pmca2/Atp2b2) in the joggle mouse, a novel mutant that displays ataxic gait by postnatal day 12. Expression of Pmca2 mRNA in the joggle mouse is only 5% of that in the wild type mouse. The insertion is located 15 bp downstream of the donor splice site of the exon containing the initiation codon. Chimeric mRNA composed of the 5'-region of Pmca2 and the IAP element were detected, indicating that some of the primary transcripts are terminated by polyadenylation signals in long terminal repeats of the IAP element. We also identified cryptic splice sites in the IAP element that are likely involved in aberrant splicing of the Pmca2 primary transcripts that leads to rapid degradation of mRNA through nonsense mediated mRNA decay. Ataxia was observed in compound heterozygous mice carrying the joggle mutation and the wriggle mutation, a previously reported missense Pmca2 mutant. Thus, we attributed ataxia in joggle mice to reduced expression of Pmca2, resulting from insertion of the IAP element.

Estimation of the time of divergence between Japanese Mishima Island cattle and other cattle populations using microsatellite DNA markers.

We applied the theory of random genetic drift to determine the divergence history of a closed cattle population over the relatively short timescale of several hundred years. The divergence history of the closed population of Mishima Island cattle, a national natural treasure of Japan, was examined, and the results were compared with historical documents. Inbreeding depression in the isolated population was investigated for body size and fertility. Twenty-one DNA microsatellite markers in Mishima Island cattle and 3 major breeds from the mainland were genotyped. For the mainland breeds, all 21 or 20 markers were segregating. However, nearly half the number of loci (9 of 21) was fixed in the Island cattle. The average number of alleles per locus of Island cattle was markedly lower than that in the mainland breeds. These results support the theory that Island cattle have been isolated for a considerable period of time. The number of generations of isolation was estimated as 14.1-22.6, and the year of divergence was calculated as 1778-1846. In view of these findings, we propose that Island cattle diverged from the mainland population at around 1800 and were isolated for about 200 years. These estimates are in agreement with historical documents showing that divergence occurred between 1672 and 1880. The total inbreeding coefficient of the present population was predicted to be in the range of 0.51-0.60. However, historical reports dated over 100 years do not support changes in fertility, so that there is no evidence for inbreeding depression.

Numerical variation of teeth in the wild house musk shrew Suncus murinus captured from Nagasaki, Japan.

Wild populations of Suncus murinus from Nagasaki were thought to be extinct, although specimens from Nagasaki are kept at the National Science Museum, Tokyo. Variation in the number of teeth, including both congenital and postnatal absence, was observed in 25 of 85 individuals. All 25 abnormal individuals were checked using a micro-CT unit to document the presence or absence of embedded teeth and any traits of postnatal absence. Four of these had congenitally absent teeth, whereas the rest showed signs of postnatal absence. There was no significant difference in the ratio of length of tooth group P(4)M(3) against palatal length between individuals with congenital absent teeth and normal ones. Because S. murinus in captivity shows a high rate of periodontal disease, we suggest that the high rate of postnatal absence of teeth in the wild population is due to oral diseases such as periodontal disease or other traumatic factors.

Tooth size variability and relevance of numerical variation in the Japanese serow.

We analysed the variability of the lower cheek teeth of the Japanese serow, Capricornis crispus, from Aichi Prefecture, Japan, based on the coefficient of variation (CV), and examined tooth size variability. The mesiodistal (MD) and buccolingual (BL) crown diameters of permanent cheek teeth were measured, and an index of the crown surface (MDxBL) was calculated. As a result, the CV for MD measurements ranged from 3.6 to 6.3, and that for BL measurements ranged from 3.6 to 6.5. In males and females, the first molar (M(1)) was the least variable (3.6-4.1) and the second premolar (P(2)) was the most variable (5.0-6.5), except for the case of BL in females. The crown surface index showed a morphological gradient that increased from mesial to distal, and the values of P(2) in both sexes were extremely small. Overall, cheek tooth variability was not influenced by developmental factors, but appeared to be inversely related to functional factors. The highest coefficient of variation was observed for P(2), which was characterised by a simple shape with a single cusp, and an extremely small size compared to other teeth. Only P(2) showed congenital absence or impaction in the lower dentition. We also confirmed the relationship between tooth size variability and numerical variation.

Quest for the cause of oligodontia in Suncus murinus (Soricomorpha, Soricidae): Morphological re-examination.

Many mammal species, including humans, exhibit oligodontia, or a lack of certain teeth in their dentition. However, the causes of the condition remain controversial. In the house musk shrew, Suncus murinus, both osteological and odontogenetic factors, which are genetically determined, have been implicated as causes of oligodontia. We examined 350 dry cranial specimens of S. murinus, representing seven laboratory strains and two wild populations. Oligodontia was observed in the upper third incisor (I(3)) and upper third premolar (P(3)); no supernumerary teeth were observed. None of the specimens with oligodontia had embedded teeth, as determined by soft X-ray. Congenital oligodontia was observed on P(3) and I(3). Moreover, the type and frequency of missing teeth differed among strains/populations. We compared the ratio of P(4)M(3) to PL as a possible cause of oligodontia. For P(3), we compared normal individuals to those with missing teeth within populations/laboratory strains, but did not detect significant differences. I(3) was compared among three laboratory strains: NAG (I(3) absent in 97%), TESS, and RYU (normal strains). TESS and RYU individuals had a longer distance from the upper fourth premolar to the upper third molar (P(4)M(3)), relative to palate length (PL), than NAG individuals. Specimens with oligodontia at I(3) and P(3) showed no difference in the ratio of P(4)M(3) length to PL. These findings suggest that osteological factors do not account for the absence of P(3) and I(3). Therefore, we believe that oligodontia in S. murinus depends on odontogenetic factors. In addition, as for I(3) and P(3)-absent, possibility related to some genes was thought about.

Changes in Ucp1, D2 (Dio2) and Glut4 (Slc2a4) mRNA expression in response to short-term cold exposure in the house musk shrew (Suncus murinus).

The house musk shrew (Suncus murinus), or so-called suncus, is a cold-intolerant mammal, but it is unclear why it is susceptible to low temperatures. Cold-intolerance may be the result of lower thermogenic activity in brown adipose tissue (BAT). The early phase of severe cold exposure is a critical period for suncus. Therefore, we exposed suncus to mildly cold temperatures (10-12 degrees C) for 1 to 48 h to increase non-shivering thermogenesis without causing stress and measured changes in the expression of uncoupling protein 1 (Ucp1), type II iodothyronine 5'-deiodinase (Dio2=D2), and glucose transporter 4 (Slc2a4=Glut4) in BAT. These mRNAs play a major role in non-shivering thermogenesis and are mainly regulated by the sympathetic nervous system via direct beta-noradrenergic innervation of BAT. During cold exposure, Ucp1 expression in BAT increased steadily over time, albeit only slightly. Neither D2 nor Glut4 expression in BAT increased immediately; however, they had increased significantly after 24 h and 48 h of cold exposure. These findings suggest that the responsiveness of mRNA regulation is weak and thus may be involved in cold-intolerance in suncus.

Mapping of jog locus to the region between D6Mit104 and D6Mit336 on mouse chromosome 6.

The joggle mouse is a recessive ataxic mutant carrying an unknown mutation in a C3H/He (C3H)-derived chromosomal segment. Taking advantage of the mouse genome database, we selected 127 DNA microsatellite markers showing heterozygosity between C3H and C57BL/6J (B6) and a first round of screening for the joggle mutation was performed on B6-jog/+ partial congenic mice (N4). We identified 4 chromosomal regions in which 13 microsatellite markers show heterozygosity between C3H and B6. Then, we analyzed the genotype of these 4 chromosomal regions in mice that showed the joggle phenotype and mapped the jog locus between markers D6Mit104 (111.4 Mb) and D6Mit336 (125.1 Mb) (an interval of 13.7 Mb) on chromosome 6. By using a partial congenic strain together with the mouse genome database, we successfully mapped the chromosomal localization of the jog locus much more efficiently than by conventional linkage analysis.

Monoclonal Suncus Antibodies: Generation of Fusion Partners to Produce Suncus-Suncus Hybridomas.

We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.

Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus).

Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. © 2016 Wiley Periodicals, Inc.