Comparative FISH mapping of human cDNA clones to chromosomes of the musk shrew (Suncus murinus, Insectivora)

Forty-one cDNA clones of human functional genes were newly mapped to chromosomes of the musk shrew (Suncus murinus, Insectivora) by fluorescence in situ hybridization, and a comparative cytogenetic map of 51 genes, including 10 genes reported in our previous study, was constructed between human (HSA) and musk shrew (SMU) chromosomes. In this comparative map, the 51 genes localized to human autosomes, except HSA 8, 16, and 20, were mapped to 15 shrew autosomes, except SMU 4, 16, 17 and 18. Twelve conserved segments were identified between human and shrew chromosomes, and six segments among the musk shrew, human, and mouse. Our results defined the presence of at least one inversion and several interchromosomal rearrangements that occurred during evolution after the two species diverged from a common ancestor. Localization of three major histocompatibility complex (MHC) genes to shrew chromosome 3 suggested that the MHC genes of the musk shrew are located in a cluster on chromosome 3. The cytogenetic map constructed in this study is the first cytogenetic map with many functional genes in insectivore species. This approach provides clues for clarifying the chromosomal evolution in this order.     

Mating stimulates estradiol production by ovaries of the musk shrew (Suncus murinus).

Asian musk shrews (Suncus murinus) are induced ovulators, but exhibit no cyclic changes in reproductive structures or in sexual behavior. Mating behavior is induced by contact with a male. To determine if mating induces changes in ovarian steroidogenesis, ovaries removed from unmated animals and at 3, 10, 15, and 36 h after mating were cultured for 4 h in the presence or absence of gonadotropins (LH + FSH, 1 microgram/ml). Histological analysis revealed no obvious changes in follicular size or appearance at the end of culture in ovaries cultured at 3 and 10 h post-mating, as compared with ovaries from unmated shrews, and mating did not stimulate any discernable changes in steroid secretion in these two groups. However, at the end of the culture period, ovulation had occurred or was occurring in ovaries from 35% of the animals ovariectomized at 15 h after mating, and corpora lutea (CLs) were present in 39% of ovarian pairs obtained 36 h after mating. At 15 h post-mating, ovaries with ovulations secreted three times more estradiol than did ovaries that showed no evidence of stimulation by mating, but there were no differences in testosterone or progesterone production. In contrast, ovaries isolated 36 h post-mating with CLs secreted dramatically more of all three steroids than ovaries without CLs (23, 13, and 52 times more estradiol, testosterone, and progesterone, respectively). These data are consistent with plasma concentrations of estradiol at the time of ovariectomy, which were twice as high at both 15 and 36 h after mating, in animals whose ovaries showed evidence of ovarian stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structure of the atrial cardiocytes in the house musk shrew (Suncus murinus).

The atrial and ventricular cardiocytes of the house musk shrew were examined by immunohistochemistry, and the right atrium containing the auricle was studied by transmission electron microscopy. The atrial natriuretic peptide (ANP)-granules of the cardiocytes in the auricle and the rest of the atrium were also analyzed by ultrastructural morphometry. On immunohistochemistry, ANP immunoreactivity was detected in the atria, with the most intensely reacting cardiocytes being localized in the right auricular part of the atrium. ANP immunoreactivity was not detected in the ventricular muscles. On ultrastructure, in most of the atrial cardiocytes including the auricle, ANP-granules, well-developed Golgi apparatus and rough endoplasmic reticulum were observed, and the nuclei were characteristically situated in the periphery of the cardiocytes, being different from many other mammalian hearts. The ANP-granules were classified into two types (A and B), with most of these granules being located in the paranuclear region in association with the Golgi apparatus, and a few ANP granules being observed throughout the sarcoplasmic layers intervening between the myofibrilar bundles. On ultrastructural morphometry, the total number of granules in the right auricular cardiocytes was significantly greater than those in the atrial cardiocytes, and the diameter of the A-granules was significantly greater than that of the B-granules in both the atrial and auricular cardiocytes.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Planned reproduction of the house musk shrew (Suncus murinus)

To investigate methods for the planned reproduction of the house musk shrew (Suncus murinus), increased reproduction was attempted from May through August, 1980 using 13 females and 13 males obtained from the breeding colony in the Laboratory of Animal Genetics, Faculty of Agriculture, Nagoya University. As a result, a reproduction colony consisting of 128 females and 60 males was formed in September, 1981. This was followed by repeated selective culling until a planned reproduction system with monthly production of over 100 pups was established in December, 1981. The production results for one year, 1982, showed that among the 994 females mated, there was a gestation rate of 74.1%, a parturition rate of 89.8%, average litter size of 3.5, a weaning rate of 77.2%, and a productive index of 1.8. A total of 1,780 weanlings was obtained and stable production throughout the year was achieved.     

Patterns of sexual receptivity in the female musk shrew (Suncus murinus)

Female musk shrews (Suncus murinus) were tested daily to examine patterns of sexual receptivity. When only mounting was used as a criterion (to avoid pregnancy), nonpregnant females remained sexually receptive to males every day for 14 consecutive days. When insemination was allowed, most females continued to copulate for the first 5 days of pregnancy. Receptivity declined markedly around Day 10 of gestation, but a few females were receptive even into late pregnancy. Lactating females copulated with males 5 and 10 days after parturition. In general, unlike most mammals studied in the laboratory, the nonpregnant female musk shrew has no behavioral estrous cycle. Musk shrews are ready to mate anytime except in mild to late pregnancy, and even then occasionally mating is found.     

Unusual features of sperm ultrastructure in the musk shrew Suncus murinus

The spermatozoon of the Musk shrew Suncus murinus displays unusual fine anatomical features. In the head, the equatorial segment of the acrosome is recessed within a waist in the sperm nucleus in a way that could afford some protection for this fusogenic region, perhaps during penetration of the egg vestments. The perinuclear material has distinctive inner and outer regions, the latter being arranged as a series of coxcomblike spikes. These structures in Suncus could serve to anchor its characteristically giant acrosome to the sperm nucleus. In the tail, small aggregations of electron dense material appear against some centriolar triplets and also, proximally, against some axonemal doublets. In the midpiece, prominent satellite material aggregates proximally over the inner border of the dense fibers. More distally, the material maintains a close relationship with and primarily lies between the corresponding axonemal doublets and dense fibers 5 and 6, the latter being positioned asymmetrically in relation to the remaining dense fibers.     

Mastication in the musk shrew,Suncus murinus (mammalia,Soricidae)

High-speed cinematography shows that Suncus murinus (Crocidurinae) masticates fast (mean 5.5, 5–10 masticatory cycles per sec). Their grasping behavior is not stereotyped. The unilateral mandibular movements combine vertical, anteroposterior, and lateral displacements; and any masticatory sequence may include crushing, repositioning, shearing, and grinding components. Size and consistency of food influence the duration of individual chewing cycles. As food is transferred to the new working side, the chewing direction reverses, either near maximum closure or near maximum opening. An unfused mandibular symphysis permits tilting movements of the two halves of the mandible. Food may be squeezed between the lower incisors. The working side tilts outward during closing; this may improve shearing or grinding action. The closing phase is posteriorly directed. Thus, the masticatory movements of these shrews differ from those that have been described in many other mammals.     

Cloning of putative uncoupling protein 1 cDNA in a cold-intolerant mammal, the house musk shrew (Suncus murinus)

The house musk shrew (Suncus murinus), or suncus, is a unique experimental animal. We recently showed that this mammal is cold intolerant and hypothesized that its sensitivity to cold is caused by low thermogenic activity in brown adipose tissue (BAT). Thermogenesis in BAT is performed by a unique mitochondrial protein, uncoupling protein 1 (UCP1). Interestingly, only eutherians possess UCP1, and some traits in the suncus resemble those in the Ucp1-ablated mouse, including cold intolerance, histology of BAT, and obesity resistance. In a previous study, we hypothesized that UCP1 may not be present in BAT of the suncus or may be dysfunctional. Therefore, we performed cDNA cloning of suncus Ucp1 and compared it to homologs from other species. The deduced amino acid sequence showed high similarity to other mammalian UCP1. Northern blot analysis revealed mRNA in BAT, as in other mammals. However, a difference in an amino acid residue was observed in an important residue for thermogenesis. Genomic sequence analysis showed that this difference existed in our two genetically distant laboratory colonies. These results suggest that cold intolerance in the suncus is derived from low thermogenic activity of UCP1 and may exist in wild house musk shrews.     

Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition.     

Growth and sexual maturation of the male house musk shrew (Suncus murinus)

The sexual maturation in the male house musk shrew, Suncus murinus, was investigated by weighing gonads, accessory sexual organs and various other organs, and measuring testosterone concentrations in the plasma from birth to 50 days of age. The histological and histochemical studies of the testis were also carried out. Testicular weight increased markedly from 10 days of age. On 15 days of age, the activity of delta 5-3 beta-Hydroxysteroid dehydrogenase (HSD) was detected in Leydig cells. In accordance with an increase of HSD activity, the concentrations of plasma testosterone raised rapidly from 15 days of age and reached a peak on Day 30. Growth of prostate glands and seminal vesicles showed a sigmoidal pattern and their significant growth began from Day 25. Sperms were detected first in the seminiferous tubules on Day 28, and most tubules were filled with many sperms on Day 33. From these results, the beginning of sexual maturation, i.e. puberty, of the male shrew is considered to be around Day 25. The weight gain of testes, accessory sexual organs and the body became slower on Day 40. The male animal of Day 40 could mate and impregnate the female. Therefore, the male shrew seems to attain to the sexual maturity between days 35 and 40.     

The force driving mating behavior in the house musk shrew (Suncus murinus)

The mating behavior of the domesticated house musk shrew was observed in detail under laboratory conditions. The observations revealed that the house musk shrew has a series of behaviors before copulation. Tactile, auditory and chemical senses appear to function as flags for the recognition of conspecifics and to promote the development of an interaction between the sexes. The tactile senses and the use of the snout were particularly important in the mating sequence, and mutual contact appeared to give rise to driving the sequence to completion. The two sexes contacted each other 'politely', came to mounting by continuous following, and the male finished with a series of post-ejaculatory offensive behaviors and scent markings. The variation in the contact reduced once the female commenced tail-wagging. The ratio of the time spent in front-and-behind contact to that spent in multi-lateral contact increased when both sexes commenced following formation. This ratio was maintained until the male's post-ejaculatory offensive behavior finished. The series of mating behavior was completed by the continuous touching of both sexes and by changes in the manner of contact.     

Gonadal influences on sexual behavior in the male musk shrew (Suncus murinus)

In this series of experiments the hormonal bases for male copulatory behavior in the musk shrew (Suncus murinus) were examined. Male musk shrews failed to show copulatory behavior after castration. Testosterone replacement fully reinstated sexual behavior. Males castrated at birth, and tested as adults after receiving testosterone implants, did not show male-typical sexual behavior. It appears that the gonads are essential for the regulation of male sexual behavior in this primitive mammal.     

Histochemical study on the external ear of Suncus murinus (Indian musk shrew)

The histochemical study of the Ear of female Suncus murinus (Indian musk shrew) was studied by the use of the cholinesterase technique. Good and sharp results were obtained while studing the AChE activity in the hair, hair follicle, hair papillae, muscle spindle, and nerve endings. The investigations were performed under constant pH, temperatures 37 degrees C to 40 degrees C, and incubation times, from 16 hours to 18 hours. The hair follicle and hair shafts showed strong positive reaction; hair papillae also showed strong positive reaction of AChE activity, while hair showed the banded appearance and the muscle spindles showed mild reaction of the AChE activity.     

Red-eyed dilution (rd), a novel coat-color mutant gene in the musk shrew (Suncus murinus, Insectivora).

A novel coat-color mutant was found in the musk shrew (Suncus murinus). Mutant shrews were characterized by light-gray coat, pinkish skin and red eyes. Mating experiment demonstrated that the mutant character was controlled by a single autosomal recessive gene. The gene could be traced back to at least four heterozygous carriers captured in Naha city, Okinawa in 1983. The name, red-eyed dilution, was proposed for this mutant character with the gene symbol rd. Linkage analysis proved no close relationship of the rd locus with the cr (cream coat color) and ch (curly hair) loci. The red-eyed dilution shrews (+/+, rd/rd) could easily be distinguished from the cream coat shrews with dark-red eyes (cr/cr, +/+) and the double homozygotes exhibiting light-cream coat with pink eyes (cr/cr, rd/rd). The rd gene has been maintained in the OKI line about at 75% of its frequency in every generation. We have started to develop a new line triple-homozygous for the cr, ch and rd genes.     

Diurnal fluctuations of heart rate, body temperature and locomotor activity in the house musk shrew (Suncus murinus).

Diurnal fluctuations of heart rate (HR), body temperature (BT) and locomotor activity (LA) in the unanaesthetized and unrestrained house musk shrew (Suncus murinus) were studied using a telemetry system. Six adult male shrews (Jic:SUN) weighing 60-70 g were used in the present study. They were housed under conditions of 24 C and a 12/12-hr light-dark cycle. HR, BT and LA were recorded over 10 days, following the post-implantation period (10 days or more) of the telemetric transmitter. A clear nocturnal rhythm of LA was shown, while intermittent and short-term LA were shown during the light period. The mean HR was 323.5 +/- 8.8 bpm in the light period and 354.3 +/- 5.2 bpm in the dark period, and the fluctuation of HR showed a nocturnal pattern. A nocturnal pattern was also observed in BT fluctuation, and all animals lowered their body temperature from 35-37 C to approximately 30 C or below, mostly during the light period. The fall of body temperature progressed over 2-3 hr, and then rose to the baseline temperature rapidly within approximately 30 min. While the body temperature fell, HR markedly decreased to approximately 100 bpm. These results suggest that the shrew has unique physiological properties in maintaining metabolic balance which are anticipated to be caused by the dramatic alteration of the autonomic nervous function.     

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.     

Fine structure of the lutein cell in the house musk shrew,Suncus murinus

Summary The ultrastructure of lutein cells during pregnancy and the post partum period was examined by transmission electron microscopy in the house musk shrew, Suncus murinus. Lutein cells on day 13 of pregnancy contained an extensive system of anastomosing tubules or cisternae of smooth ER and many enlarged mitochondria with numerous tubulovesicular cristae. From day 13 on, the number of small granules, possibly lysosomes, increased gradually. Between day 20 and 25, the loss of smooth ER began and elongated, or flattened mitochondria increased. Regressing lutein cells observed after parturition were characterized by abundant large dense bodies, bizarre mitochondria and a decrease in the amount of smooth ER. Unusual forms of mitochondria were always observed after day 5 of pregnancy. Two types could be distinguished; one, found frequently in the second third of pregnancy, was ring-, disc-, cup-or dumb-bell-shaped with tubulovesicular cristae, and the other, found exclusively in the last third of pregnancy and after parturition, was elongated, flattened and sometimes twisted. The paucity of lipid droplets was a characteristic feature of the lutein cells of this species. The significance of these ultrastructural changes of cellular organelles is discussed in relation to the ovarian and plasma levels of progesterone.     

Development and characterization of CATS markers for genetic linkage mapping in the house musk shrew, Suncus murinus.

To serve as an initial step in developing an ideal genetic marker map for the house musk shrew, Suncus murinus, 318 comparative anchor tagged sequence (CATS) primer pairs were assessed for polymorphism ascertainment and linkage mapping. Of the 112 (35.2%) CATS primer pairs that were successfully amplified by PCR in the shrew, 18 (16.1%) showed polymorphism between two mutant strains, BAN-kc, oeb and WZ. Linkage analysis of the polymorphic CATS markers and three visible mutant genes, kc, oeb and wz, genotyped in a 77 F2 mapping panel from a cross of the two mutant strains, assigned wz and five CATS markers into three linkage groups. Sequence analysis revealed that two (ADA and TXN) out of nine CATS amplified sequences had a total of six deletions of varying sizes and 17 single nucleotide polymorphisms (SNPs). BLAST search identified three CATS (ADA, CYP1A2, and TXN) products matching the genes from which they were originally designed, while the remaining six markers could not be identified. Together with the use of the detected SNPs as genetic markers, the five CATS markers linkage mapped in this species will serve as anchors in establishing the first framework map for locating loci affecting all heritable qualitative and quantitative traits in the musk shrew.