Epithelial cell proliferation and apoptosis in the developing murine palatal rugae

Epithelial cell proliferation and apoptosis during morphogenesis of the murine palatal rugae (PR) were examined histochemically by using anti-bromodeoxyuridine (BrdU) and the terminal deoxynucleotidyl transferase-mediated UTP nick-end-labelling (TUNEL) technique. Formation of the PR rudiment was observed as an epithelial placode in fetuses at 12.5 days post-coitus (dpc). During the PR formation, BrdU-positive cells were detected mainly in the epithelium of the interplacode and interprotruding areas in fetuses administered BrdU maternally at 2 h before killing. TUNEL-positive cells were detected only at the epithelial placode area in 12.5-14.5 dpc. At 16.5-18.5 dpc, the BrdU-positive cells were decreased in number in the epithelial cells at the interprotruding area of the PR. Only a few TUNEL-positive cells were observed in the protruding area of the PR at 16.5 dpc. These results suggest that cell proliferation and apoptosis in the palatal epithelium are involved spatiotemporally in the murine PR morphogenesis.     

Gross morphological,histological and scanning electron specifications of the oropharyngeal cavity of the hooded crow (Corvus cornix pallescens)

The present study was carried out on the oropharyngeal cavity of the hooded crow to investigate the gross and microscopic structures via gross anatomy, light microscopy and scanning electron microscopy (SEM). The gross anatomy clarified the elongated triangular shape of the oropharyngeal cavity with a non-protruding tongue with a bifid apex. The lingual body contained median groove rostrally and separated caudally from the root by a transverse papillary crest. The laryngeal mound located posterior to the lingual root, contained midline laryngeal cleft and bounded caudally by a transverse row of pharyngeal papillae. The palate contained choanal cleft rostrally and infundibular slit caudally in addition to five palatine ridges. By light microscopy, the dorsal lingual epithelium was highly keratinised stratified squamous with a lingual nail in the most rostral part of the apex. Then, the thickness of the keratin layer decreased caudally, while in the ventral surface, the lining epithelium became non-keratinised. The entoglossum supported the lingual body and root, but not extended to the apex. The lining epithelium of the palate was also keratinised stratified squamous and became none-keratinised at the oral side of the choanal cleft. There were numerous lobules of polystomatic salivary glands in the lingual root and the palate. SEM revealed the arrangement of different types of papillae covering both the floor and the roof of the oropharynx besides numerous openings of salivary glands in the lingual root, laryngeal mound and the palate. These findings reflect the functional relationship of the oropharyngeal cavity of the hooded crow during feeding.     

Effects of EGF receptor ligands on fetal ovine myoblasts

Epidermal growth factor (EGF) receptors are widely distributed in mammalian tissues, including muscle. One ligand of these receptors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is also strongly expressed in adult muscle. However, in vitro studies of EGF action in cultured muscle cells of different species have yielded conflicting results. The purpose of this study was to investigate the potential role of EGF and related factors in the growth and development of fetal ovine muscle. High affinity EGF receptors were detected on clonally purified ovine fetal myoblasts, using [(125)I] human EGF as a ligand (K(d) values of 47 and 54 pM in separate experiments). Competitive binding studies in mixed secondary cultures showed that EGF had the highest affinity for the fetal ovine receptor, followed by HB-EGF and transforming growth factor alpha (TGF-alpha). These ligands all stimulated DNA synthesis in clonally purified ovine myoblasts, with their relative potencies at 0.1 nM reflecting their receptor binding affinities. Maximal effects were seen at 1-10 nM. EGF (10 nM) did not significantly inhibit the differentiation of clonally purified fetal ovine myoblasts, although there was increased proliferation of nondifferentiating cells. Hence a variety of EGF receptor ligands have the potential to influence the proliferation ovine muscle cell precursors in utero, but it is unlikely that they promote differentiation.     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Über die frühfetale Entwicklung der Dünndarm-Mukosa des Rindes (Bos primigenius taurus).

The development of the bovine small intestine was examined in 24 embryos and fetuses by light microscopic, scanning and transmission electron microscopic methods. Special reference was paid to the genesis of the epithelium and particularly of the villi intestinales. The primitive intestine consists of one layer of epithelial cells surrounded by mesenchym and tunica serosa. The fetal intestine (up to the 24th week of gestation) shows all the morphologic structures of the adult. In small intestine the development of cryptae and villi intestinales starts before the 7th week of gestation and progresses with a proximo-distal gradient. Epithelial proliferation that gives rise to primary epithelial villi makes epithelium become temporarily stratified. Finger-like secondary villi develop by proliferation of the mesenchym. In addition to this process mucosal folds occur in duodenum giving rise to villi by segmentation. At the same time the differentiation of epithelium starts. The fetal small intestine, like many other fetal tissues displays masses of glycogen.     

Surgical repair of a cleft soft palate in an alpaca

A 26-day-old entire male alpaca was referred for surgical treatment of a cleft soft palate. Mandibular symphysiotomy and three-layer closure of the palatal defect resulted in primary intention healing. Despite complications related to anaesthesia the alpaca made a full recovery.     

Effects of growth factors on development of fetal islet B-cells in vitro

To investigate the role of growth factors (epidermal growth factor [EGF], betacellulin, and activin A) in the development of islet B cells of rat fetal pancreatic explants in vitro, pancreases from rat fetuses at day 18 of gestation were cultured for 96 hr, with or without these growth factors. Culture medium was changed every 24 hr, and the level of insulin released in the culture medium was measured. After 72 hr of culture, pancreases were examined histologically. As a result, EGF promoted cell proliferation, but reduced B cell volume. Whereas, betacellulin and activin A inhibited cell division, but promoted increased B cell volume and insulin secretion, especially activin A, which stimulated insulin release in a time dependent manner. These results suggest that EGF, betacellulin, and activin A promote pancreatic cell proliferation, islet B-cell differentiation, and islet B-cell differentiation and functional maturation, respectively, and that EGF, betacellulin, and activin A, in this order, regulate islet B-cell neogenesis.     

Application of a temporary palatal prosthesis in a puppy suffering from cleft palate

A 3-month-old Schnauzer was presented with congenital defects of the secondary palate. On the clinical examination, coughing, sneezing, drainage of nasal discharge from the external nares and poor growth were found. Vital signs and results of blood examination were within normal ranges. Thoracic radiography revealed mild pneumonia in the right lung lobes. In a puppy suffering from cleft palates, a palatal prosthesis was applied to the hard palate in order to protect the surgical wound, because a routine surgery was not successful. A palatal prosthesis was applied and held in place using the instant glue and plastic bands to protect the surgical wound following the third repeated surgery. Although a small oronasal fistula still remained, there was no functional defect. This prosthesis was easy to apply and helpful to protect the surgical wound. In addition, this implant could be placed or adjusted without or sedation/anesthesia.     

YAP对梅花鹿鹿茸间充质细胞增殖与分化的影响

以梅花鹿为研究对象,通过流式细胞术、荧光定量PCR等方法,探讨YAP(Yes-associated protein,即Yes相关蛋白)对鹿茸间充质细胞增殖与分化的影响.分离培养鹿茸间充质细胞,添加一定浓度的YAP抑制剂Verteporfin后,通过流式细胞术检测发现,细胞的增殖活性被显著抑制,G0/G1期细胞百分比增加...     

Effects of concurrent exposure to 3-methylcholanthrene and vitamin A on fetal development in rats

To investigate the effect of the environmental pollutants, polycyclic aromatic hydrocarbons (PAHs), on retinoic acid-induced teratogenesis, all-trans-retinoic acid (RA) dissolved in corn oil (120 mg/kg) was administered orally to pregnant rats at the 11th day of gestation with and without the prior intraperitoneal treatment with 10 mg/kg 3-methylcholanthrene (3-MC) for 3 days. Dams were killed on the 20th day of pregnancy. The examinations of fetuses revealed that 3-MC barely enough to cause induction of P-450 in pregnant dams had profound embryo-toxic effects: the fetal resorption amounted to approximately 60% of total number of implantations. The fetuses survived weighed less than the control fetuses. All of RA-treated mothers had fetuses with abnormalities, and the main malformations were absence of tail (100%), caudal and sacral malformations (100%), and cleft palate (42%). Pregnant dams received both 3-MC and RA had a reduced severity of tail anomaly (33%), while the rest, 67%, had short vestigial tail. Caudal and sacral malformations were detected but at a milder degree. We did not observe cleft palate in this group. The concurrent treatment of dams with 3-MC and RA led to an increased inducibility of cytochrome P-450 and subsequently, CYP1A1 dependent enzyme activity higher than those observed after the injection of 3-MC alone. UDP-glucuronyl-transferase activity was also markedly induced in concurrent 3-MC and RA group higher than that in 3-MC alone. We suggest that the induction of P-450 and alteration of metabolic enzyme activities may play an important role in reducing the teratogenic potency of RA. However, RA-treatment did not retard the embryo-toxic effect of 3-MC but rather potentiated.     

Distribution of apoptotic cells and apoptosis-related molecules in the developing murine palatine rugae

Distribution of apoptotic cells and expression of the apoptosis-related factors p53, bcl-2 and bad during morphogenesis of the murine palatine rugae (PR) were examined histochemically using the terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) technique and specific antibodies against apoptosis and cell cycle-related molecules. Formation of the PR rudiment was controlled by cell proliferation and apoptosis in the palatal epithelium. TUNEL-positive cells were detected only at the epithelial placode area at 12.5-13.5 days post coitus (dpc), but only a few cells were positive at the protruding PR area at 14.5-16.5 dpc. Bcl-2 protein was expressed mainly in the areas outside of those containing TUNEL-positive cells at 15.5 -6.5 dpc. P53 protein was not detected throughout gestation. Bad was detected in the epithelial layer at 13.5 and 15.5 dpc and overlapping the apoptotic area at 13.5-15.5 dpc. Apoptosis of palatal epithelial cells might therefore involve spatiotemporally regulated expression of bad during murine PR development.     

转GFP基因核移植牛胚胎的研究

试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20 ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著（P>〖JP2〗0.05）;mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。     

EGF和FGF-2对猪皮下脂肪神经嵴干细胞增殖和分化的影响

【目的】探究表皮生长因子（epidermal growth factor，EGF）和成纤维细胞生长因子2（fibroblast growth factor 2，FGF-2）对猪皮下脂肪神经嵴干细胞（neural crest stem cells，NCSCs）增殖及分化的影响，以优化猪皮下脂肪神经嵴干细胞的培养条件。【方法】通过体外分离培养原代猪皮下脂肪NCSCs，免疫荧光染色鉴定NCSCs标志物p75 NTR，并用不同浓度的EGF和FGF-2（0和0、10和10、10和20、20和10、20和20、30和30 ng/mL）作用于传代猪皮下脂肪NCSCs，用CCK-8试剂盒测定细胞增殖率，确定细胞生长的最适EGF和FGF-2浓度，将试验分为空白组和最适浓度组，测定两组细胞的生长曲线，成脂化诱导后油红O染色，对比两组细胞的脂滴生成量。【结果】免疫荧光染色结果显示，原代猪皮下脂肪NCSCs经p75 NTR鉴定呈阳性。CCK-8细胞增殖试验结果显示，EGF和FGF-2的浓度均为20 ng/mL时对传代猪皮下脂肪NCSCs的促增殖作用最佳。生长曲线显示，两组细胞均在第5~9天处于对数生长期，第10~15天细胞增殖减缓，逐渐到达停滞期。油红O染色结果显示，最适浓度组胞质内的脂滴生成量远多于对照组。【结论】在培养液中添加20 ng/mL EGF和20 ng/mL FGF-2对猪皮下脂肪NCSCs的增殖和分化有促进作用。     

A mixed epithelial and stromal tumor of the kidney in a ringtail lemur (Lemur catta)

Primary renal tumors are rare neoplasms in nonhuman primates. This report describes a mixed epithelial and stromal tumor of the kidney (MESTK) in a 14.5-year-old female ringtail lemur. The well-demarcated, solid, and cystic mass was located in the pelvis of the left kidney and consisted histologically of both epithelial and mesenchymal components. The mesenchymal cells were arranged in fascicles around cysts lined by a well-differentiated epithelium. Neither the mesenchymal nor the epithelial parts showed significant nuclear atypia or mitotic figures. To our knowledge, only 1 similar case, classified as adenoleiomyofibromatous hamartoma, has been reported in a ringtail lemur. In humans this tumor affects predominantly perimenopausal women and can express estrogen and progesterone receptors. However, neither estrogen nor progesterone receptors could be identified by immunohistochemistry in the tumor of the present ringtail lemur. Therefore, a hormonal mechanism could not be demonstrated in this case.     

Reduced birth weight,cleft palate and preputial abnormalities in a cloned dog

The aim of the present study was to report a novel developmental abnormality in a cloned dog. A fibroblast cell line was established from an 8-year-old male German shepherd dog. In vivo matured oocytes were retrieved from a large breed dog, and the nucleus was removed from each oocyte. A donor cell was injected into an enucleated oocyte, and the oocyte-cell couplet was fused electrically. After chemical activation, the resulting embryos were transferred into a naturally estrus-synchronized recipient dog, and two cloned pups were delivered by Cesarean section 60 days later. One cloned pup (Clone 1) was healthy, but the other (Clone 2) had a birth weight of only 320 g and cleft palate, failure of preputial closure at the ventral distal part, and persistent penile frenulum. Clone 2 was raised by stomach feeding until Day 40 after birth, where palatoplasty was performed. The abnormalities in external genitalia in Clone 2 resulted in persistent penile extrusion that was surgically corrected. This complex developmental abnormality has not been reported in dogs previously.     

Metastasizing oral squamous cell carcinoma in an aged pig

A 10-year-old, female, pot-bellied pig (Sus scrofa) experienced a 3-month history of reduced appetite, dysphagia, and weight loss. Clinical examination revealed a mass in the left part of the oral cavity extending from the hard to the soft palate. At necropsy, a firm, white, poorly demarcated ulcerated mass at the left hard and soft palate with metastases to the left retropharyngeal lymph node and the lung was observed. Additional findings included a uterine adenocarcinoma, a hepatocellular adenoma, and nodular hyperplasias in spleen and adrenal glands. Histologically, the poorly demarcated, infiltrative growing oral mass consisted of islands, cords, and single epithelial cells with moderate squamous differentiation. Cells were strongly positive for cytokeratin by immunohistochemistry. Similar cells were found in the left retropharyngeal lymph node and the lung. The present findings represent the first report of a metastasizing oral squamous cell carcinoma in a pig.     

汞的生殖毒性和发育毒性

本文综述了汞对雄性及雌性动物的生殖毒性 ,汞的胎盘及哺乳毒性、汞对胚胎和出生后动物发育的影响。汞能降低雄性鼠类及鹌鹑的交配能力 ,破坏鸭的睾丸间质细胞及精母细胞 ,并能破坏禽类的蛋壳结构而影响其生殖功能。汞不仅能通过胎盘途径 ,而且能经过哺乳途径进入胎儿体内 ,从而引起神经损伤等病变。汞能抑制蝌蚪的正常变态 ,引起动物胚胎被吸收或胚胎死亡 ,也可以导致胎儿腭裂、四肢缺失及脑部畸形。接触甲基汞的雄性小鼠对其第二代仔鼠的发育毒性比第一代更显著。     

Use of a Cortico-Cancellous Bone Graft in the Repair of a Cleft Palate in a Dog

We report the successful use of a cortico-cancellous bone graft to repair a cleft of the secondary palate in a 6-month-old Akita dog. The cleft extended from the incisal papilla to the posterior border of the soft palate. It was 10 mm wide in the hard palate and 16 mm in the soft palate. The cleft was repaired using the "push-back" method with single-pedicled mucoperiosteal flaps to close the cleft in the soft tissues, and a free bone graft from the tibia (cortex with cancellous bone attached) to fill the defect in the hard palate. Two and one-half years after the operation the graft was indistinguishable from surrounding bone on radiographs. Maxillary growth appeared to have progressed normally, resulting in normal occlusion.     

Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Effect of replacement of pyruvate/lactate in culture medium with glucose on preimplantation development of porcine embryos in vitro

The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.