Effect of Mesenchymal Cells on Myoblast Sheets Embedded in Collagen Gel.

The objective of this study was to investigate the effects of mesenchymal cells on myoblasts in long-term cultivation of myoblast cell sheets. Sheets of myoblasts and mesenchymal cells from Japanese rabbit oral mucosa were generated and analyzed by histochemistry, Western blot, and reverse transcription-polymerase chain reaction. The presence of desmin and type IV collagen, which is seen in normal muscle tissue, was also confirmed in all the sheets produced. Expression of desmin and type IV collagen showed a decrease under co-culture conditions. In addition, expression of genes important in maintaining the undifferentiated state (Pax7, CD34, myogenin, MyoD) in myoblasts was observed throughout the long cultivation period. Insulin-like growth factor was expressed only when the mesenchymal cells were co-cultured with myoblasts. These data suggest that the presence of mesenchymal cells in a long-term co-culture system influences myoblast differentiation.

Midline sensory nerve supply to the anoscrotal junction: a study using human male fetuses.

The origin of the posterior scrotal nerve is considered to be the bilateral pudendal nerves but the course to the midline is still obscure. Using 5 late-stage human male fetuses, we identified the single nerve through the intramuscular midline septum of the bulbospongiosus and the bilateral nerves along the left and right sides of the septum. Thus, the posterior scrotal nerve showed a variation: a single midline trunk or bilateral nerves. Branches of the bilateral pudendal nerves ran medially between the muscle and Cowper's gland and, at the midline area, they joined or associated closely. During the proximal course, much or less, the nerve penetrated the superior part of the muscle. The nerve entered the subcutaneous tissue at and near the perineal raphe. The communication with intrapelvic autonomic nerves were suggested behind Cowper's gland. Notably, the midline skin immediately anterior to the anus carried a considerable dense supply of thin sensory nerves. However, these nerves seemed to come from a space between the rectal smooth muscle and the external anal sphincter, not from the posterior scrotal nerve. Therefore, surgical treatment of the intersphincteric layer was likely to injure the original sensory nerve supply to the anterior anal skin.

Histological study of the developing pterygoid process of the fetal mouse sphenoid.

The pterygoid process undergoes ossification of both the cartilage and membrane. However, few studies have attempted to explore the sequential development of the pterygoid process. Using histological examination, we performed morphological observations of the pterygoid process and surrounding tissue. ICR mice at embryonic days 13.5-18.0 and postnatal day 0 were used for morphological observations of the pterygoid process. By embryonic day 14.5, a mesenchymal cell condensation forming the anlage of the future medial pterygoid process differentiated into osteoid-like tissue and cartilage. At embryonic days 15.5-16.5, cartilage cells were clearly evident in the medial pterygoid process. In the medial pterygoid process, a bone collar was evident and calcified bone tissue surrounded the cartilage. At this point, a mesenchymal cell condensation formed the anlage of the pterygoid hamulus. At embryonic days 17.0-18.0, the cartilages were located along the lower and posterior border of the medial pterygoid process. A metachromatically stained matrix first became detectable around cells located in the pterygoid hamulus. On the other hand, at embryonic day 13.5, a metachromatically stained matrix was already evident in the space between the flattened cells in the lateral pterygoid process. At embryonic day 17.0, a hypertrophic cell zone had clearly formed in the diaphysis. On the basis of our present investigation, the lateral pterygoid process can be classified as primary cartilage, whereas the medial pterygoid process can be classified as secondary cartilage. Furthermore, it was found that the pterygoid hamulus is formed latest in the medial pterygoid process.

Is the ultimobranchial body a reality or myth: a study using serial sections of human embryos.

Reported morphologies of the ultimobranchial body had varied between researchers: a cluster of mitotic cells, a duct-like structure and a rosette-like cell mass. To clarify the true morphology, we studied tilted horizontal sections of 20 human embryos (crown-rump length 5-18 mm; 4-6 weeks). The sections displayed a ladder-like arrangement of the second to fourth endodermal pouches and, in 5 early embryos we found the fifth pouch attached to the fifth ectodermal groove near the fourth pharyngeal arch artery. The bilateral fifth pharyngeal pouches protruded anterolaterally to form a U-shaped lumen surrounding the arytenoid swelling. The third to fifth pouches were each characterized by a pedal-shaped inferior end. We identified several types of cell clusters as candidates for the ultimobranchial body, but morphologically most of them were, to various degrees, likely to correspond to the blind end of the lower pouch when cut tangentially. Because of the topographical relation to the common carotid artery, a cyst-like structure with a cell cluster seemed to be the most likely candidate of the ultimobranchial body (a common anlage of the thymus and parathyroid). However, we were not able to deny a possibility that a certain plane cutting the pouch end incidentally provided such a cyst-like structure in sections. At any stage, the ultimobranchial body might not appear as a definite structure that is discriminated from others with routine staining. A concept of the ultimobranchial body might be biased by comparative anatomy that shows the ultimobranchial gland in adult birds and reptiles.

Fetal development of the minor lung segment.

The mediobasal segment (S7) of the right lung has been considered to correspond to the cardiac lobe generally seen in mammals. To investigate fetal development of the right mediobasal segmental bronchus (B7), we examined paraffin-embedded serial sections of 15 embrynic and fetal lungs at 7-8 weeks (serial sections) as well as semiserial sections of 8 fetuses at 15-18 weeks (semiserial sections). All of the smaller specimens did not contain B7, but 2 of the 8 larger specimens carried B7: one was found in the immediately anterior side of the inferior pulmonary vein, while in the other, the subdivisions (B7a, B7b) were overriding the vein. Although the incidence might be underestimated because of observations using semiserial sections, the B7 was most likely to develop secondarily during a period from 8 to 15 weeks. Fetal topographical changes (mainly, the descent) of the middle lobe and the inferior pulmonary vein might relate with the secondarily budding of B7. The present result does not reduce a clinical relevance of B7 as a segmental bronchus of the lung segment system.

Expression of carbonic anhydrase in the fetal eye and extra-ocular tissues.

Carbonic anhydrases (CAs) plays a critical functional role in the ciliary body and retina for maintenance of microenvironment. With immunohistochemistry using orbital contents from 8 human fetuses (12-16 weeks of gestation), we examined expressions of CAs isozymes-1, 2, 3, 6, 7 9 and 12 and found strong reactivity of CA9 in extra-ocular fibrous tissues in the anterior and posterior eyes. CA9 is known to express in the fetal joint cartilage to maintain pH against hypoxia: actually, in the present specimens, the SO pulley and its tendon was strongly positive for CA9. The CA9-positive anterior fibrous tissues were positive for smooth muscle actin and connected the orbital aspect of the 4 rectus muscle with the palpebral conjunctiva, whereas the posterior tissue was negative for smooth muscle actin and corresponded to the lateral insertion tendon of the orbitalis muscle. The anterior CA9-positve tissues seemed to correspond to the primitive form of the sleeve and pulley system. Any of matrix substances (collagen types I and II, aggrecan, versican, fibronectin, tenascin and hyaluronan) displayed a distribution pattern specific for the CA9-positive fibrous tissues. Therefore, whether or not CA9 was positive in the fibrous tissue seemed not to depend on the tissue components such as the extracellular matrix and intermediate filaments but to suggest a stressful condition such as hypoxia, unsuitable base balance and/or under mechanical stress.