Motoki Sasaki

Prostaglandin F2α increases endothelial nitric oxide synthase in the periphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis

Prostaglandin F(2)(alpha) (PGF(2)(alpha)) released from the uterus causes alterations in luteal blood flow, reduces progesterone secretion, and induces luteolysis in the bovine corpus luteum (CL). We have recently discovered that luteal blood flow in the periphery of the mature CL acutely increases coincidently with pulsatile increases in a metabolite of PGF(2)(alpha) (PGFM). In this study, we characterized changes in regional luteal blood flow together with regional alterations in endothelial nitric oxide synthase (eNOS) expression during spontaneous luteolysis and in response to PGF(2)(alpha). Smooth muscle actin-positive blood vessels larger than 20 microm were observed mainly in the periphery of mature CL. PGF(2)(alpha) receptor was localized to luteal cells and large blood vessels in the periphery of mid-CL. PGF(2)(alpha) acutely stimulated eNOS expression in the periphery but not in the center of mature CL. Injection of the NO donor S-nitroso-N-acetylpenicillamine into CL induced an acute increase in luteal blood flow and shortened the estrous cycle. In contrast, injection of the NOS inhibitor l-NAME into CL completely suppressed the acute increase in luteal blood flow induced by PGF(2)(alpha) and delayed the onset of luteolysis. In conclusion, PGF(2)(alpha) has a site-restricted action depending on not only luteal phase but also the region in the CL. PGF(2)(alpha) stimulates eNOS expression, vasodilation of blood vessels, and increased luteal blood flow in periphery of mature CL. Furthermore, the increased blood flow is mediated by NO, suggesting that the acute increase in peripheral blood flow to CL is one of the first physiological indicators of NO action in response to PGF(2)(alpha).

Comparative Functional Morphology of the Masticatory Apparatus in the Long‐snouted Crocodiles

The masticatory muscles and their related structures of the skull were observed in the Indian gavial (Gavialis gangeticus), the false gavial (Tomistoma schlegelii), and the African slender‐snouted crocodile (Mecistops cataphractus) to detail some morphological differences in comparison with the other crocodile species, and to compare and elucidate the functional strategy of themasticatory apparatus in these long‐snouted species. The Musculus pterygoideus posterior was relatively smaller in the three species compared with many short‐snouted crocodiles. It suggests that the masticatory power in fish‐eating long‐snouted species is not so high as in the short‐snouted crocodiles, while the masticatory muscles were morphologically different among the three long‐snouted species as follows. The M. pterygoideus posterior of the false gavial was extended in the lateral side of the lower jaw unlike the Indian gavial. The M. pseudotemporalis and the Fenestra supratemporalis were largely developed in the Indian gavial, however we suggest that the other two species possess the weak bundles in this muscle. The false gavial and the African slender‐snouted crocodile have the pterygoid bone well‐developed extending dorso‐ventrally and it is suggested that the M. adductor mandibulae posterior attached to the pterygoid bone may be much larger than the Indian gavial. These data morphologically clarify the masticatory mechanism in the long‐snouted crocodiles different from the short‐snouted species, and demonstrate that the evolutional strategy to share the functional role in the masticatory muscles have been differently established between the Indian gavial and the other two species. We also obtained the morphological data in the fossil skull of the Machikane crocodile (Toyotamaphymeia machikanense) and concluded from the fossil characters that the considerable developments of the M.pterygoideus posterior and the M.pseudotemporalis in this species had not morphologically been consistent with both the Indian and false gavials.

Expression and localization of apelin and its receptor APJ in the bovine corpus luteum during the estrous cycle and prostaglandin F2alpha-induced luteolysis.

Angiogenesis, changes in blood flow, and extracellular matrix remodeling are the processes associated with the development and demise of the bovine corpus luteum (CL) during the estrous cycle. APJ (putative receptor protein related to angiotensin type 1 receptor) is a G-protein-coupled receptor, and its ligand, apelin, has been identified as a novel regulator of blood pressure and as an angiogenic factor. We hypothesized that the apelin-APJ system is involved in luteal function. This study investigated whether apelin-APJ exists in bovine CL and determined their expression profiles and localization during luteal phase and prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced luteolysis. During the luteal phase, apelin mRNA expression increased from early to late CL and decreased in regressed CL. APJ mRNA expression increased from early to mid-CL and remained elevated in late and regressed CL. Apelin and APJ proteins were immunohistochemically detected only in the smooth muscle cells of intraluteal arterioles during the luteal phase. PGF(2)(alpha) stimulated apelin and APJ mRNA expression at 0.5-2 and 2 h respectively, and then the mRNA expression of apelin-APJ was inhibited from 4 h during PGF(2)(alpha)-induced luteolysis. Additionally, apelin mRNA and protein were stimulated at 1 h after PGF(2)(alpha) injection only in the periphery of mid- but not early CL. The present study indicated that the apelin-APJ was localized in the smooth muscle cells of intraluteal arterioles, and responded to PGF(2)(alpha) at the periphery of mid-CL in the cow. Thus, the apelin-APJ system may be involved in the maturation of CL and the luteolytic cascade as a regulator of intraluteal arterioles in cow.

Absence of carotid rete mirabile in small tropical ruminants: implications for the evolution of the arterial system in artiodactyls.

The intracranial carotid rete (or rete mirabile epidurale) is a unique blood vascular system supplying the brain of artiodactyls, which have either an involuted or no internal carotid artery. Although the lesser and greater mouse deer (Tragulus javanicus and T. napu, respectively) are ruminants, the rete mirabile epidurale is absent. In these animals, as in non‐artiodactyls, such as canines, equines and humans, the complete internal carotid artery supplies the brain. It is currently uncertain whether the absence of the rete is confined to mouse deer among ruminants. The absence of the rete in mouse deer provides new insights into the evolution of the arterial system in artiodactyls.

Carpal bone movements in gripping action of the giant panda (Ailuropoda melanoleuca)

The movement of the carpal bones in gripping was clarified in the giant panda (Ailuropoda melanoleuca) by means of macroscopic anatomy, computed tomography (CT) and related 3‐dimensional (3‐D) volume rendering techniques. In the gripping action, 3‐D CT images demonstrated that the radial and 4th carpal bones largely rotate or flex to the radial and ulnar sides respectively. This indicates that these carpal bones on both sides enable the panda to flex the palm from the forearm and to grasp objects by the manipulation mechanism that includes the radial sesamoid. In the macroscopic observations, we found that the smooth articulation surfaces are enlarged between the radial carpal and the radius on the radial side, and between the 4th and ulnar carpals on the ulnar side. The panda skilfully grasps using a double pincer‐like apparatus with the huge radial sesamoid and accessory carpal.

Immunohistochemical Study on the Distribution and Relative Frequency of Endocrine Cells in the Stomach of the Malayan Pangolin, Manis javanica

The distribution and relative frequency of six kinds of endocrine cells in the stomach of the Malayan pangolin, Manis javanica were studied immunohistochemically using the avidin–biotin–peroxidase complex method. The stomach of the pangolin has three regions of mucous gland, one oxyntic gland and one pyloric gland. Cells immunoreactive for chromogranin, serotonin, somatostatin, BPP and glucagon were detected in all of the gastric glands, while gastrin‐immunoreactive cells were found in the entire gastric gland except for the oxyntic gland. The distribution pattern of endocrine cells in the mucous gland and pyloric gland was mainly from the middle to apical portions of the glands. The endocrine cells were rare or not detected in the basal portion of all of the mucous glands and pyloric gland, but they were also found in the basal portion of the oxyntic gland. The distribution pattern of the endocrine cells in the mucous and pyloric glands suggested that this position facilitates a quick response to the luminal ingesta. The wide distribution of gastrin‐immunoreactive cells in all of the mucous glands and pyloric gland was the most remarkable finding. This distribution suggests a major function of gastrin‐immunoreactive cells for the digestive process in the Malayan pangolin stomach.

Expression of prostaglandin F2α (PGF2α) receptor and its isoforms in the bovine corpus luteum during the estrous cycle and PGF2α-induced luteolysis.

Prostaglandin F2α (PGF2α) induces luteolysis via a specific receptor, PTGFR. Although PTGFR mRNA expression in the bovine corpus luteum (CL) has been studied previously, changes in PTGFR protein and its localization are not fully understood during the life span of the CL. In addition to full-length PTGFR, several types of PTGFR isoforms, such as PTGFRα (type I) and PTGFRζ (type II), were reported in the bovine CL, suggesting isoform-specific luteal action. Full-length PTGFR mRNA in the bovine CL increased from the early to the mid-luteal phase and decreased during luteolysis, whereas PTGFR protein remained stable. PTGFR protein was localized to both luteal and endothelial cells and was expressed similarly during the life span of the CL. Like full-length PTGFR mRNA, PTGFRα and PTGFRζ mRNA also increased from the early to mid-luteal phases, and mRNA of PTGFRζ, but not PTGFRα, decreased in the regressing CL. During PGF2α-induced luteolysis, the mRNAs of full-length PTGFR, PTGFR,α and PTGFRζ decreased rapidly (from 5 or 15 min after PGF2α injection), but PTGFR protein decreased only 12 h later. Silencing full-length PTGFR using small interfering RNA prevented PGF2α-stimulated cyclooxygenase-2 (PTGS2) mRNA induction. By contrast, PGF2α could stimulate vascular endothelial growth factor A (VEGFA) mRNA even when full-length PTGFR was knocked down, thus suggesting that PGF2α may stimulate PTGS2 via full-length PTGFR, whereas VEGFA is stimulated via other PTGFR isoforms. Collectively, PTGFR protein was expressed continually in the bovine CL during the estrous cycle, implying that PGF2α could function throughout this period. Additionally, the bovine CL expresses different PTGFR isoforms, and thus PGF2α may have different effects when acting via full-length PTGFR or via PTGFR isoforms.

Radial sesamoid bone as a part of the manipulation system in the lesser panda (Ailurus fulgens)

The well-developed radial sesamoid bone presented a rod-like shape in the lesser panda. It could be separated into two components: (1) an ulnar cartilaginous, (2) a radial osseous part. The radial sesamoid bone was connected with four elements as follows: (1) the tendon of the M. abductor pollicis longus, (2) M. abductor pollicis brevis and M. opponens pollicis, (3) Aponeurosis palmaris, and (4) Flexor retinaculum. The bone made no articulation with the first metacarpal. The movement of the radial sesamoid bone may be controlled by the connecting muscles and muscle-related structures. It is suggested that the bone acts as a supporting ridge in the gripping action in the lesser panda. However, we suggest that the grasping mechanism is obviously different from that of the giant panda, in which the radial sesamoid bone is connected strongly with the first metacarpal.

A preliminary study on origin of Callosciurus squirrels introduced into Japan

ABSTRACT To examine the origin of Callosciurus erythraeus introduced to Japan, we compared mitochondrial DNA control region sequences of eight haplotypes from Japan with those of 42 haplotypes from Taiwan. There were two distinct phylogroups in Japan. Six haplotypes from Japan were included in the Taiwan population, suggesting that they could have been introduced from Taiwan. Two haplotypes, however, were distantly related to the cluster consisting of all Taiwanese haplotypes and six Japanese haplotypes. The uncorrected genetic distances (5.2–5.7%) between these two haplotypes and the out-group (Callosciurus finlaysonii from Laos) were less than those (8.3–9.8%) between these two haplotypes and the cluster consisting of the other six Japanese haplotypes and all Taiwanese haplotypes. Both C. erythraeus and C. finlaysonii have variable external characteristics, such as pelage color, and include many variable subspecific forms, so that it is difficult to evidently identify both species on the basis of their external characteristics. Callosciurus finlaysonii may have been also introduced in Japan with both species now coexisting in Japan.

A Lectin Histochemical Study on the Testis of the Babirusa, Babyroussa babyrussa (Suidae)

The distribution of lectin bindings in the testis of babirusa, Babyrousa babyrussa (Suidae) was studied histochemically using 10 biotinylated lectins, Peanut agglutinin (PNA), Ricinus communis agglutinin (RCA I), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Soybean agglutinin (SBA), Wheat germ agglutinin (WGA), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA), Concanavalin A(Con A) and Ulex europaeus agglutinin (UEA I). Nine of 10 lectins showed a variety of staining patterns in the seminiferous epithelium and interstitial cells. The acrosome of Golgi‐, cap‐ and acrosome‐phase spermatids displayed various PNA, RCA I, VVA, SBA and WGA bindings, indicating the presence of glycoconjugates with d‐galactose, N‐acetyl‐d‐galactosamine and N‐acetyl‐d‐glucosamine sugar residues respectively. No affinity was detected in the acrosome of late spermatids. LCA, PSA and Con A which have affinity for d‐mannose and d‐glucose sugar residues were positive in the cytoplasm of spermatids and spermatocytes. DBA was positive only in spermatogonia. In addition to DBA, positive binding in spermatogonia was found for VVA, WGA and Con A, suggesting the distribution of glycoconjugates with N‐acetyl‐d‐galactosamine, N‐acetyl‐d‐glucosamine, d‐mannose and d‐glucose sugar residues. Sertoli cells were stained intensely with RCA I, WGA and Con A. In Leydig cells, RCA I and Con A were strongly positive, while WGA, LCA and PSA reactions were weak to moderate. The present findings showed that the distribution pattern of lectin binding in the testis of babirusa is somewhat different from that of pig or other mammals reported previously.