Kisspeptin neurons mediate reflex ovulation in the musk shrew (Suncus murinus).

The present study investigated whether kisspeptin-G protein-coupled receptor 54 (GPR54) signaling plays a role in mediating mating-induced ovulation in the musk shrew (Suncus murinus), a reflex ovulator. For this purpose, we cloned suncus Kiss1 and Gpr54 cDNA from the hypothalamus and found that suncus kisspeptin (sKp) consists of 29 amino acid residues (sKp-29). Injection of exogenous sKp-29 mimicked the mating stimulus to induce follicular maturation and ovulation. Administration of several kisspeptins and GPR54 agonists also induced presumed ovulation in a dose-dependent manner, and Gpr54 mRNA was distributed in the hypothalamus, showing that kisspeptins induce ovulation through binding to GPR54. The sKp-29-induced ovulation was blocked completely by pretreatment with a gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons to induce ovulation in the musk shrew. In addition, in situ hybridization revealed that Kiss1-expressing cells are located in the medial preoptic area (POA) and arcuate nucleus in the musk shrew hypothalamus. The number of Kiss1-expressing cells in the POA or arcuate nucleus was up-regulated or down-regulated by estradiol, suggesting that kisspeptin neurons in these regions were the targets of the estrogen feedback action. Finally, mating stimulus largely induced c-Fos expression in Kiss1-positive cells in the POA, indicating that the mating stimulus activates POA kisspeptin neurons to induce ovulation. Taken together, these results indicate that kisspeptin-GPR54 signaling plays a role in the induction of ovulation in the musk shrew, a reflex ovulator, as it does in spontaneous ovulators.

The complete control of murine pregnancy from embryo implantation to parturition.

The ovary is the main secretory source of progestin and estrogen and is indispensable to the maintenance of all events of pregnancy in mice. The purpose of this study was to control all processes of pregnancy in mice, from embryo implantation to parturition, without ovaries. The ovaries were removed before embryo implantation, and a single injection of medroxyprogesterone acetate (MPA) was given. Embryo implantation was induced by leukemia inhibitory factor, which can substitute 17ß-estradiol (E(2)). Continuous exposure to E(2) was necessary at mid-pregnancy, when placentation was completed. All mice sustained pregnancy without ovaries before parturition, which was initiated by the removal of E(2) and MPA. Murine pregnancy is a complicated process involving embryo implantation, placentation, and parturition. Complete control of pregnancy was achieved with the simple treatment of MPA and E(2) after induction of embryo implantation. Here, time-dependent events in the uterus during pregnancy could be realized without the ovaries, because the initiation of each event could be stringently controlled by hormonal treatments.

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Immunolocalization of aquaporin-4 in the brain, kidney, skeletal muscle, and gastro-intestinal tract of chicken.

Aquaporin-4 (AQP4), a member of the water channel family, has high water permeability and multi-functional potentiality. Although an avian AQP4 homolog has recently been identified, its overall localization is still largely unknown. This study demonstrates the presence of AQP4 in several organs of chicken by using a specific chicken AQP4 antibody. Western blot analysis has revealed two bands of chicken AQP4 (30 and 32 kDa) in the brain, proventriculus, pectoral muscle, kidney, and ureter. The brain is the primary expression site of AQP4 in chicken. Immunohistochemical analysis of the brain has shown the highest AQP4 immunoreactivity around the cerebral ventricles, blood vessels, and the Purkinje cells. In peripheral organs, AQP4-immunoreactive elements have been observed in the ureter, glandular cells of the proventriculus, sarcolemma of the pectoral muscle, and the epithelium of the ceca and the rectum. Moreover, a heavily stained network of AQP4-immunoreactive fibers has been detected within the enteric plexuses.

Embryo implantation is blocked by intraperitoneal injection with anti-LIF antibody in mice.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

Genetic diversity of 21 experimental chicken lines with diverse origins and genetic backgrounds.

The genetic characteristics and diversity of 21 experimental chicken lines registered with the National BioResource Project of Japan were examined using mitochondrial D-loop sequences and 54 microsatellite DNA markers. A total of 12 haplotypes were detected in the 500-bp mitochondrial DNA sequences of the hypervariable segment I for 349 individuals of 21 lines. The 12 haplotypes belonged to three (A, D, and E) haplogroups, out of the eight (A‒H) common haplogroups in domestic chickens and red junglefowls. The haplogroups A and D were widely represented in indigenous chickens in the Asian and Pacific regions, and the haplogroup E was the most prevalent in domestic chickens. Genetic clustering by discriminant analysis of principal components with microsatellite markers divided 681 individuals of 21 lines into three groups that consisted of Fayoumi-, European-, and Asian- derived lines. In each of the cladograms constructed with Nei's genetic distances based on allele frequencies and the membership coefficients provided by STRUCTURE and with the genetic distance based on the proportion of shared alleles, the genetic relationships coincided well with the breeding histories of the lines. Microsatellite markers showed remarkably lower genetic heterozygosities (less than 0.1 observed heterozygosity) for eight lines (GSP, GSN/1, YL, PNP, BM-C, WL-G, BL-E, and #413), which have been maintained as closed colonies for more than 40 years (except for #413), indicating their usefulness as experimental chicken lines in laboratory animal science research.

Encephalomyocarditis virus is potentially derived from eastern bent-wing bats living in East Asian countries.

Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

Localization of sympathetic, parasympathetic and sensory neurons innervating the distal ileum of the cattle.

After oral challenge of the pathological prion protein, the causative agent of bovine spongiform encephalopathy, the pathogen was first detected in the distal ileum and then deposited in the brain. The present study aims determining the possible neuronal transporting pathways from the ileum to the brain in the cattle using a tracer protein. After horseradish peroxidase was injected into the wall of the distal ileum in the calf, almost all labeled neurons were detected in the celiac and cranial mesenteric ganglion complex. Only a few labeled neurons existed in the caudal mesenteric ganglion and the paravertebral ganglia. They were sympathetic postganglionic neurons. In the dorsal root ganglia T5 to L4, some sensory neurons were found to be labeled. Only a small number of parasympathetic preganglionic neurons were labeled in the dorsal motor nucleus of the vagus nerve. No labeled sensory neurons were found in the nodose ganglion. These results suggest that the pathological prion protein is mainly transported to the spinal cord and brain via the sympathetic nervous system and partially via the sensory neurons in the dorsal root ganglia. The vagus nerve does not seem to contribute to the transport of the pathogen from the ileum directly.

Seasonal variation in the number of deaths in Pteropus lylei at Wat Pho Bang Khla temple, Thailand.

Flying foxes have been widely studied as they are well-known reservoirs of infectious agents. Understanding their population dynamics might help to explain seasonal patterns of disease prevalence, and contribute towards the conservation of flying fox populations. Therefore, this study explored the annual variation in the number of deaths in P. lylei. The study was conducted from 2015-2017, at a Buddhist temple in Thailand, which is the roosting site of P. lylei. The average total number of bat deaths in a month significantly varied between times of a year. A peak was observed during March and May, which ranged in the period of birthing and lactating. There were no significant differences in the average total number of bat deaths in a month between sexes or age classes across times of a years.

Nighttime behavioral study of flying foxes on the southern coast of West Java, Indonesia.

Flying foxes are important in the maintenance of forests and diversity. However, knowledge of their behavioral ecology, especially of movement and foraging patterns, which are essential for conservation and management of their populations, are not well known. Therefore, movement behavior of two individuals of Pteropus vampyrus were examined using an Argos telemetry system, and foraging pattern of Pteropus spp. was directly observed, at West Java province, Indonesia in October 2017. The maximum distance between the location at which bats were released and their furthest roost, recorded via satellite telemetry, was approximately 100 km. This reflects the long-distance flight ability of P. vampyrus. Daytime roosting sites and nighttime foraging places consisted of several types of habitats, such as intact forests, agricultural lands, and residential areas. This evidence indicated that there was habitat overlap between humans and bats in West Java province. According to direct observation of the behaviors of flying foxes at two locations within residential areas, various activities such as wing spreading, excretion, fighting, aggressive calls, movement, hanging relax, and hanging alert were found. The number of bat-visits to the trees varied among night hours, and had a positive correlation with the number of fruit dropping. The data obtained in this study have improved our understanding of nighttime behavior and habitat utilization of P. vampyrus, that can be used to support landscape management, species conservation, and disease prevention in regions of Southeast Asia.

A novel method of gene transduction to the murine endometrium using in vivo electroporation.

To investigate the molecular pathways involved in successful embryo implantation in mammals, we developed a novel method for gene transduction into the murine endometrium using in vivo electroporation. Plasmid DNA with an enhanced green fluorescence protein (EGFP) gene was injected into the uterine cavity of non-pregnant female mice, and electrical pulses were subsequently applied to the uterine horn using plate electrodes. EGFP expression was found only in the uterine luminal epithelium (LE), but not in the stroma. EGFP fluorescence in the LE was limited to the site where the positive side of the electrodes was placed during electric stimulation. These results demonstrated that our novel method enabled us to transduce a gene into a desired location of the murine uterus.

Diurnal behavior and activity budget of the golden-crowned flying fox (Acerodon jubatus) in the Subic bay forest reserve area, the Philippines.

Acerodon jubatus (the Golden-Crowned flying fox) is an endemic species in the Philippines, which was suspected to be a host of the Reston strain of the Ebola virus. As nocturnal animals, the flying foxes spend daytime at the roosting site, which they use for self-maintenance and reproduction. To understand the variation in diurnal behavior and time allocation for various activities in the Golden-Crowned flying fox, we investigated their daytime behavior and activity budget using instantaneous scan sampling and all occurrence focal sampling. Data collection was performed from 07:00 to 18:00 hr during January 8-17, 2017. The most frequent activity was sleeping (76.3%). The remaining activities were wing flapping (5.0%), self-grooming (4.2%), hanging relaxation (3.4%), wing spread (2.9%), movement (2.4%), mating/courtship (2.4%), aggression (1.9%), hanging alert (1.2%), excretion (0.1%) and scent marks (0.05%). The frequency of sleeping, wing flapping, self-grooming, hanging relaxation, aggression, mating/courtship and movement behaviors changed with the time of the day. Females allocated more time for resting than males, while males spent more time on the activities that helped enhance their mating opportunities, for example, movement, sexual activity and territorial behavior.

The Window of Implantation Is Closed by Estrogen via Insulin-Like Growth Factor 1 Pathway.

BACKGROUND: The opening and closing of the implantation window is important for successful pregnancy in eutherians. The recent study demonstrated that the window of uterine receptivity was prepared by the sole action of progesterone in mice, but the mechanism to close the window remained to be elucidated. METHODS: The pregnant mice were ovariectomized on the evening on the third day of pregnancy with a single injection of medroxyprogesterone acetate to induce delayed implantation (DI). Several treatments were applied to DI mice. The uterine receptivity after treatment was assessed by examining cell proliferation in the uterine luminal epithelium (LE). The gene expressions in the endometrium were investigated by RNA-seq. The p<0.05 was considered significant. RESULTS: Cell proliferation in the LE ceased only when the window of implantation was open. Estrogen (E2) stimulated cell proliferation in the LE rendered the uterus refractory. The high throughput gene expression analysis by RNA-Seq showed that the insulin-like growth factor 1 (IGF1) pathway was the candidate to close the implantation window under E2. In vivo administration of IGF1 to delayed implantation mice resulted in proliferation in the LE cells. CONCLUSION: This study demonstrated that the window of uterine receptivity was closed by E2, which was mediated by the IGF1 pathway.

The genetic diversity of D-loop sequences in eastern bent-winged bats (Miniopterus fuliginosus) living in Wakayama Prefecture, Japan.

The eastern bent-winged bat (Miniopterus fuliginosus) is an insectivorous bat that lives in the caves, throughout Japan [11]. The bats aggregate in cave in populations of tens to thousands of individuals. We examined the mitochondrial D-loop sequences of bats in Wakayama, Japan, and divided them into 35 haplotypes. The sequences of 3 haplotypes in Wakayama were the same as those of 10 Miniopterus fuliginosus individuals living in China. Given the substitution rate of the D-loop region, we speculated that the bats had moved between Japan and China within the last 16,000 years. We could not determine how the bats crossed the sea; however, it is possible that the bats undergo dynamic movement widely throughout East Asia.

Daytime behavior of Pteropus vampyrus in a natural habitat: the driver of viral transmission.

Flying foxes, the genus Pteropus, are considered viral reservoirs. Their colonial nature and long flight capability enhance their ability to spread viruses quickly. To understand how the viral transmission occurs between flying foxes and other animals, we investigated daytime behavior of the large flying fox (Pteropus vampyrus) in the Leuweung Sancang conservation area, Indonesia, by using instantaneous scan sampling and all-occurrence focal sampling. The data were obtained from 0700 to 1700 hr, during May 11-25, 2016. Almost half of the flying foxes (46.9 ± 10.6% of all recorded bats) were awake and showed various levels of activity during daytime. The potential behaviors driving disease transmission, such as self-grooming, mating/courtship and aggression, peaked in the early morning. Males were more active and spent more time on sexual activities than females. There was no significant difference in time spent for negative social behaviors between sexes. Positive social behaviors, especially maternal cares, were performed only by females. Sexual activities and negative/positive social behaviors enable fluid exchange between bats and thus facilitate intraspecies transmission. Conflicts for living space between the flying foxes and the ebony leaf monkey (Trachypithecus auratus) were observed, and this caused daily roosting shifts of flying foxes. The ecological interactions between bats and other wildlife increase the risk of interspecies infection. This study provides the details of the flying fox's behavior and its interaction with other wildlife in South-East Asia that may help explain how pathogen spillover occurs in the wild.

Morphological characteristics of tectal neurons of layer I projecting to the nucleus geniculatus lateralis ventralis in chick.

Some visual information is sent to the nucleus geniculatus lateralis ventralis (GLv) via the cells in layer I (I cells) of the tectum in birds and is used for color vision, papillary reflex, and kineoptic functions. To reveal the morphological features of 'I cells' projecting to the GLv, they were retrogradely labeled with DiI (1,1'-dioctadecyl-3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate) in chicks. Two different types of neurons, 'spear dendritic I cells' and 'forked dendritic I cells' were identified. The former had small spindle-like soma and an apical dendrite extending to the tectal surface, and the latter had somewhat larger triangular or polygonal soma and plural ascending dendrites. Most of the labeled dendritic endings bifurcated horizontally in layer F, and showed the ending patterns similar to the terminals of optic nerve fibers.

The contribution of leukemia inhibitory factor (LIF) for embryo implantation differs among strains of mice.

Despite of the claim that maternal leukemia inhibitory factor (LIF) - a member of interleukin 6 (IL6) family of cytokines - plays indispensable roles for murine embryo implantation, these roles remain undefined in humans because the potency of LIF on implantation appears to vary among individuals. Here, we showed that the contribution of LIF for murine implantation was dependent on the strains of mice (ICR, C57BL/6J (B6), ddY, BALB/c, DBA/2Cr and MF1 strains). Inhibition of LIF during the implantation period caused severe disruption of embryo implantation in B6 and MF1 strains. Implantation was partly disrupted in other strains, but some embryos were implanted successfully. We speculated that other IL6 family members compensate for LIF actions on implantation in ICR, ddY, BALB/c, and DBA/2Cr strains. Indeed, the expression level of Ctf1 was upregulated by blockage of LIF function. CT-1 (encoded by Ctf1) treatment induced successful implantation without LIF in delayed implantation mice (ICR and B6) via phosphorylation of the signal transducer and activator of transcription 3 (STAT3) in the uterine luminal epithelium. Simultaneous inhibition of LIF and CT-1 did not block implantation completely in ICR mice, indicating that embryo implantation in this strain was robustly protected by LIF, CT-1 and other potential STAT3 activators. The present study might provide an explanation for the individual variation in the potency of LIF for embryo implantation in humans.

Differences in extrinsic innervation patterns of the small intestine in the cattle and sheep.

After oral challenge of the pathological prion protein, the pathogen was first detected in the distal ileum and then deposited in the brain. The present study aims determining the possible neuronal transport pathways from the small intestine to the brain in the cattle and sheep using a tracer protein. After horseradish peroxidase was injected into the wall in the duodenum of the calf and lamb and in the ileum of the lamb, the greater part of labeled neurons was detected in the celiac and cranial mesenteric ganglion complex. In the dorsal root ganglia T3 to L4 of both animals, some sensory neurons were always found to be labeled. Some parasympathetic preganglionic neurons were labeled in the dorsal motor nucleus of the vagus nerve after injections into the duodenum of the cattle and sheep, but extremely a small number of them were labeled after ileal application. The number of labeled sensory neurons in the nodose ganglion after duodenal injections of the sheep was much greater than that after duodenal application of the cattle. After ileal injections in the sheep, practically no labeled sensory neurons were found in the nodose ganglion. These results suggest that the pathological prion protein is mainly transported to the spinal cord and brain via the sympathetic nervous system and partially via the sensory neurons in the dorsal root ganglia. The vagus nerve seems to contribute to the transport of the pathogen not from the ileum, but from the duodenum.

Spinal nerve innervation to the sonic muscle and sonic motor nucleus in red piranha, Pygocentrus nattereri (Characiformes, Ostariophysi).

The red piranha, Pygocentrus nattereri, produces sounds by rapid contractions of a pair of extrinsic sonic muscles. The detailed innervation pattern of the sonic muscle of the red piranha was investigated. The sonic muscle is innervated by branches (sonic branches) of the third (S3so), fourth (S4so), and fifth (S5so) spinal nerves. The average total number of nerve fibers contained in the right sonic branches (n = 5; standard length, SL, 71-85 mm) was 151.8 (standard deviation, SD, 28.3). The occipital nerve did not innervate the sonic muscle. The sonic motor nucleus (SMN) in the piranha was identified by tracer methods using wheat germ agglutinin-conjugated horseradish peroxidase; labeled sonic motor neurons were only observed on the side ipsilateral to the sonic muscle injected with the tracer. In the transverse sections, the labeled sonic motor neurons were located in the dorsal zone (mainly large and medium neurons) and in the ventral zone (mainly small neurons) of the ventral horn. In the horizontal sections, the labeled neurons formed a rostrocaudally elongated SMN from the level of the caudal part of the second spinal nerve root to the intermediate region between the fifth and sixth spinal nerve roots. The average number of the labeled neurons (n = 5; SL, 64-87 mm) was 152.6 (SD, 7.3). We conclude that the sonic muscles of the piranha are innervated by approximately 300 sonic motor neurons located only in the spinal cord.

Distribution patterns of immunocompetent cells in the pregnant mouse uteri carrying allogeneic mouse and xenogeneic vole embryos.

Mouse and vole embryos were allogeneically and xenogeneically transferred into pseudopregnant CD-1 and immunodeficient (scid) female mice, and we investigated the distribution of immunocompetent cells, uterine natural killer (uNK) cells, mast cells and macrophages, in the implantation sites on days 6, 7 and 8 of gestation. The survival rate of the vole embryos decreased gradually with increased gestation, but the rate was higher in the scid uteri than in the CD-1 mice. The number of uNK cells increased markedly at the mesometrial triangle and the outer decidual area in the CD-1 uteri containing vole embryos; by contrast, scid uteri having vole embryos showed almost the same number as those having mouse embryos. Mast cells were present in large numbers at the myometrium, but rarely in the decidua in all types of pregnant uteri. Cells at the myometrium were more numerous in xenogeneic than in allogeneic transfer. Many mast cells appeared in the inner decidua where xenogeneically transferred vole embryos were dead and aborted. Macrophages were present in the outer decidua and myometria in all types of pregnant uteri, and their distribution pattern did not change even in aborted uterine sites. These results suggest: (1) the response of macrophages to dead embryos is completely inhibited, (2) uNK cells and mast cells increase near dead and aborted embryos, and (3) the increment in uNK cells responding to xenogeneic embryos is suppressed in scid mice, and the suppression may contribute partly to survival of the embryos.