Three Dimensional Architecture of Collagen Fibrils in the Corpus cavernosum of the Crab-eating Monkey

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1 Okajimas Folia Anat. Jpn., 73(4): , October, 1996 Three Dimensional Architecture of Collagen Fibrils in the Corpus cavernosum of the Crab-eating Monkey By Takashi NAKANO Department of Anatomy, Aichi Medical University, Nagakute, Aichi , Japan -Received for Publication, May 10, Key Words: Penis, Tunica albuginea, Trabecula, Scanning electron microscope, Cell maceration method Summary: The three-dimensional architecture of the collagen fibrils in the corpus cavernosum of the crab-eating monkey was studied by scanning electron microscope, after digestion of cellular and some extracellular elements by NaOH. The tunica was made up of undulating bundles of collagen fibrils which were arranged in inner circular, middle intermingled and outer longitudinal layers. The trabecula appeared as numerous cylindrical tubes which had once accommodated individual smooth muscle fibers. The outer surface of the trabecula was covered with a sheath woven from twisting bundles of collagen fibrils. These architectural features of the collagen fibrils seem to be related to fluctuations in the diameter and shape of the corpus during erection and detumescence. During erection, the corpus cavernosum becomes enormously enlarged, and the intracavernous pressure can greatly exceed systolic blood pressure (Carati et al., 1987; Purohit and Beckett, 1979). Therefore, the tunica albuginea surrounding the corpus requires not only elasticity but also rigidity. Strand-like trabeculae, which penetrate inward to delimit labyrinthine anastomosing cavernous sinuses, have to be slenderized and elongated to cause consequent distension of the sinuses during erection. The tunica as well as the trabeculae consist mainly of the collagen fibers with a few elastic fibers. In order to lead an appropriate hypothesis on the mechanism of penile erection, investigations of the collagen fibrils in the corpus seem important. However, there is still some uncertainty regarding the architecture of the collagen fibers due to their three-dimensional complexity, which stands in contrast to many available publications on the penile vascular system. The NaOH cell-maceration method (Ohtani, 1987) is very effective in removing cellular elements as well as some extracellular elements, making it possible to observe directly the three-dimensional architecture of collagen fibrils. Kurosawa et al. (1993) first induced the method into the human corpus, but could not obtain sufficient findings. Nakano (1995a, b) revealed clearly the three-dimensional architecture of collagen fibrils in the mouse corpus. The investigations on mammals other than the mouse must be of interest, because mechanism of erection seems to be various among different species of mammals (Fujita and Shirai, 1989). The crab-eating monkey was selected in this study, because the corpus of monkey is often used as a suitable model for the study on the mechanism of erection. It is hoped that this study will contribute to a further understanding of the intricate mechanism of penile erection. Materials and Methods Penes of three adult crab-eating monkeys, Macaca irus, bred in the laboratory animal research center in the Aichi Medical University, were used in this study. After removal of the skin, the penes were cut into several sections. For the LM study, the specimens were fixed in Zenker's fluid, dehydrated in a graded ethanol series, and embedded in paraffin. They were cut serially into transverse and longitudinal sections of 311,m and Azan stain was applied. For the SEM study, the specimens were immediately fixed with 2.5% glutaraldehyde in phosphate buffer for 2 hours and rinsed in distilled water. Then, the specimens were processed by NaOH cellmaceration method (Ohtani, 1987); they were immersed in a 10% NaOH solution for 4 days at room temperature, and rinsed in distilled water until they became transparent. After immersion in 1% tannic acid for 2-3 hours, they were rinsed in distilled water for several hours, postfixed with 2% osmium tetroxide for 1 hour, dehydrated in a graded ethanol, and dried with t-butyl alcohol freeze-drying method 185

2 186 T. Nakano (Inoue and Osatake, 1988). The specimens were cut longitudinally and transversely to the long axis of the corpus with a blade under a dissecting microscope, coated with gold and observed under a Jeol JSM 6100 scanning electron microscope. Results Light-microscopic findings The tunica albuginea surrounding the corpus cavernosum was made up almost entirely of thick collagen fibers. From the tunica albuginea finger-like trabeculae penetrated inward. In the cross sections, they appeared as rounded or irregularly polygonal profiles around the slit-like cavernous spaces. The trabeculae were predominantly composed of smooth muscle fibers which were partly separated from the cavernous spaces by collagen fibers (Fig: 1). In longitudinal sections of the corpus, the trabeculae appeared as thin strands which were generally arranged along the long axis of the corpus. The trabeculae consisted mainly of bundles of smooth muscle fibers which were oriented along the long axis of the trabeculae. The collagen fibers running along the trabecula showed a winding course (Fig. 2). Scanning electron microscopic findings After NaOH treatment, cellular elements such as smooth muscle fibers as well as some extracellular elements were completely removed without any apparent damage to collagen fibrils. Tunica albuginea Each corpus cavernosum was surrounded by a thick capsule, the tunica albuginea (about 800 1,200 um in thickness) (Fig. 3). The tunica was composed of thick bundles of collagen fibrils which took an undulating course. In the inner layer the bundles were arranged circularly, while in the outer layer then ran longitudinally along the long axis of the corpus (Fig. 4). In the region between the inner and outer layers, the circular bundles intei mingled with the longitudinal ones (Fig. 4, 5). That is, the collagen bundles were arranged in three layers (inner circular, middle intermingled and outer longitudinal). In the median plane, the grouped collagen bundles extended inward from the inner circular layer of the tunica to form the median septum (Fig. 3). Each bundle was composed of densely packed collagen fibrils running in parallel. Further, small bundled as well as single collagen fibrils ran in various directions to form the fine networks enmeshing the bundles (Fig. 5). The bundles showed a wide range of widths (about 5-25 pm) and thicknesses (about gm). Trabecula The trabecula, after NaOH treatment, extended inward from the inner circular layer of the tunica (Fig. 3). At higher magnifications, the trabecula displayed a honeycomb appearance on crosscut surface across its long axis (Fig. 6, 7). Each hole in the appearance is surrounded by a wall consisting of collagen fibrils (Fig. 7). On longitudinally cut sections, the trabeculae were arranged along the long axis of the corpus, and were frequently bifurcated and connected with adjacent trabecula to form a complex network (Fig. 8). At higher magnifications, they exhibited numerous cylindrical tubes, occupied by individual muscle fibers prior to the NaOH treatment (Fig. 9). The wall of the tube consisted of single and small-bundled collagen fibrils. The small-bundled fibrils themselves took a twisting course and gave off much thinner bundles and even single fibrils, both of which formed a finer meshwork. These fibrils of one wall frequently extended to and formed part of the neighboring wall. Such collagenous walls varied in density; the meshwork was dense and complicated at some sites but sparse in others (Fig. 9). The outer surface of the trabeculae was covered with a collagenous sheath composed mainly of thick bundles of collagen fibrils, running generally in longitudinal or oblique direction to the long axis of the trabecula. The bundles took a wavy course, often giving off thinner branches and interwound in complex fashion (Figs. 10, 11). The bundles were enmeshed by a delicate lacework knitted together by smallbundled as well as single collagen fibrils. Between the bundles were delicate networks of entangled collagen fibrils, which derived from the lacework around the bundles (Fig. 11). On the basis of the findings described above, a schematic illustration of the architecture of the collagen fibrils and the smooth muscle fibers in the corpus is presented in figure 12a. Discussion Tunica albuginea In the corpus cavernosum, it has been reported that in man the intracavernous pressure during erection can greatly exceed systolic blood pressure to become the corpus rigid (Kano et al., 1986; Carati et al., 1987; Purohit and Beckett, 1979). Therefore, the tunica albuginea surrounding the corpus has to resist possible rupture of the corpus during erection and intercourse. The tunica is made up almost entirely of thick bundles of collagen fibrils. According to widely read textbooks on human histology (Fawcett, 1986; Geneser, 1986; Ito, 1987; Fujita and Fujita, 1992), the collagen bundles are arranged in inner

3 Collagen Fibrilar Architecture in the Corpus cavernosum 187 circular and outer longitudinal layers. However, it is difficult to determine the precise arrangement of the collagen fibrils from thin sections, and threedimensional observation by SEM is indispensable. Nakano (1995a, b) examined three-dimensionally the mouse tunica treated with the NaOH cellmaceration method, and revealed that the bundles in the tunica were arranged in inner circular and outer longitudinal layers. In the present study, the bundles in the tunica of the crab-eating monkey were arranged in three layers. In the inner layer they ran circularly, while in the outer one they were longitudinal. In the middle layer, the circular bundles and the longitudinal ones intermingled. The inner circular layer serves to resist the considerable increase in diameter of the corpus during erection, while the outer longitudinal one resists lengthening. The middle layer may function to coordinate the movement of the inner circular and outer longitudinal layers during erection and detumescence. In this study, it was clear that the collagen bundles took a wavy course in the flaccid state. Such bundles seem to have stretchability until they become straight, with the enlargement of the cavernous sinuses during erection. The change in arrangement of the collagen bundles during erection is speculated on in figure 12b. During erection the tunica appears to be stretched so as to become increasingly thinner and stiffer than in its flaccid state. Trabecula During erection, the trabeculae are slenderized with consequent engorgement of the cavernous spaces. Which component of the trabeculae does relate to their slenderizing? At light microscopic level, the trabeculae in the mouse were mainly constituted of collagen bundles with a few smooth muscle fibers (Nakano, 1995a). After the NaOH cellmaceration method, the mouse trabeculae consisted of grouped collagen bundles which ran along their meshwork taking a winding course. Considered from these findings, Nakano (1995a) suggested in the mouse that the collagen bundles mainly related to changes in the diameter of the trabeculae during erection; i.e., when the tunica is extended, the collagen bundles in the trabeculae appear to be stretched until they become straight, causing a slenderizing of the trabeculae. In the crab-eating monkey, the findings were extremely different from those in the mouse. At the light microscopic level, the bulk of the trabeculae was made up of smooth muscle fibers, which were arranged in bundles running along the longer axis of the trabeculae. It is suggested that the relaxation of the smooth muscle fibers contributed to a slenderizing of the trabeculae with consequent distention of the cavernous spaces during erection (Fig. 12b). These findings may provide further support for the description by Cormack (1987) that relaxation of the smooth muscle fibers in the human trabeculae is a factor contributing to distension of the cavernous spaces. Further, contraction of the smooth muscle fibers appear to cause a narrowing of the cavernous spaces with consequent emptying of the spaces during detumescence. After NaOH treatment, the trabeculae of the crab-eating monkey exhibited numerous cylindrical tubes which had once accommodated individual smooth muscle fibers. The wall of the cylindrical tube, which is woven from twisting collagen fibrils, is considered to be highly flexible and to comply with a functional need; i.e., the smooth muscle fibers may be allowed a certain freedom of movement during erection and detumescence. It is noteworthy that collagen fibrils of one wall extended into and formed part of the neighboring wall; i.e., the smooth muscle fibers are tied together by the collagenous wall to form the bundle. This finding suggests that the collagenous wall provides a structure suitable for the coordinated movement of muscle fibers. In the mouse, the outer surface of the trabeculae was covered with a sheath woven from twisting collagen fibrils (Nakano, 1995a, b). It was suggested that the sheath may be highly flexible during erection and detumescense (Nakano, 1995a). In the monkey, however, the outer surface of the trabeculae was covered with a sheath woven from thick collagen bundles, which generally ran along the longer axis of the trabeculae taking a wavy course. Such bundles appear to have an ability to be stretched during erection. The change in arrangement of the collagen bundles during erection is speculated in figure 12b: When the smooth muscle fibers are relaxed to cause a slenderizing of the trabeculae, the stretched collagen bundles contribute to resisting the considerable lengthening of the trabeculae. It further appears that during detumescence, the collagen bundles may regain their twisting course as the smooth muscle fibers may regain their former tonus. References 1) Carati CJ, Creed KE and Keogh EJ. Autonomic control of penile erection in the dog. J Physiol 1987; 384: ) Cormach DH. Ham's Histology. pp (Lippincott, Philadelphia 1987). 3) Fawcett DW. A Textbook of Histology. pp (Saunders, Philadelphia 1986). 4) Fujita H and Fujita T. Textbook of Histology Part 2 (in Japanee). pp (Igaku Shoin, Tokyo 1992). 5) Fujita T and Shirai M. Mechanism of penile erection (in Japanese). J Clin Exp Med 1989; 148: ) Geneser F. Textbook of Histology. pp (Munksgaard, Copenhagen 1986).

4 188 T. Nakano 7) Inoue T and Osatake H. A new drying method of biological specimens for scanning electron microscopy: The t-butyl alcohol freeze-drying method. Arch Histol Cytol 1988; 51 : ) Ito T. Histology (in Japanese). pp (Nanzando, Tokyo 1987). 9) Kano K, Hanyu S and Iwanaga T. Mechanism of erection based on penile vascular organization (in Japanese). Biomed SEM 1986; 15: ) Kurosawa T, Goto Y and Banya Y. Collagen fibers in the tunica albuginea of human penis. Its arrangement and possible function in the mechanism of erection (in Japanese with English abstract). J Iwate Med Ass 1993; 45: ) Nakano T. Three-dimensional architecture of collagen fibrils in the mouse corpus cavernosum penis. Acta Anat 1995a; 152: ) Nakano T. Collagen fibrillar arrangement in corpus cavernosum/corpus spongiosum of mice. Assist Reproduct Technol Androl 1995b; 8: ) Ohtani 0. Three-dimensional organization of the connective tissue fibers of the human pancreas. A scanning electron microscopic study of NaOH treated tissues. Arch Histol Jpn 1987; 50: ) Purohit RC and Beckett SD. Penile pressures and muscle activity associated with erection and ejaculation in the dog. Am J Physiol 1979; 231: Explanation Plate of Figures I Fig. 1. Light micrograph showing the crosscut surface of the corpus cavernosum. The trabeculae (T) consisting mainly of smooth muscle fibers are partly separated from the cavernous spaces by collagen fibers (arrows). Red blood cells are seen in the cavernous spaces (asterisk). Azan stain. x300. Fig. 2. Light micrograph showing the longitudinally cut surface. The trabeculae (T) are made up mainly of smooth muscle fibers which are oriented along the long axis of the trabeculae. The collagen fibers (arrows) run along the trabecula, taking a winding course. Red blood cells are seen in the cavernous spaces (asterisk). Azan stain. x300. Fig. 3. Scanning electron micrograph showing the cross section of the penis. The corpora cavernosum (Cc) are covered with thick tunica albuginea (arrowheads). Arrow = median septum. x 25. Fig. 4. Higher magnification of a cross section of the tunica. It is clear that each collagen bundle takes an undulating course. The collagen bundles are arranged in inner circular (C) and outer longitudinal (L) layers. In the middle layer, the circular bundles (arrows) intermingle with the longitudinal ones (arrowheads). x200. Fig. 5. Higher magnification of the middle intermingled layer of the tunica. Arrow = circular bundle. Arrowheads = longitudinal bundles. x 2,500.

5 Collagen Fibrilar Architecture in the Corpus cavemosum 189 Plate I

6 190 T. Nakano Plate II Fig. 6. Cross section of the trabeculae. The trabeculae (T) form a complex framework around the cavernous spaces (asterisk). x 100. Fig. 7. Higher magnification of a cross section. The trabecula displays a honeycomb appearance. Each hole (arrowheads) in this appearance is surrounded by a collagenous wall (arrows). Asterisk = cavernous space. x 1,000. Fig. 8. A longitudinally cut section of the trabeculae. The trabeculae (T) are arranged longitudinally, and are bifucated and connect with adjacent trabecula. Asterisk = cavernous space. C = inner circular layer of the tunica. x 100. Fig. 9. Higher magnification of a longitudinally cut section. The trabecula exhibits cylindrical tubes (t). The wall (arrows) of the tube is woven from collagen fibrils which take a twisting course. x 1,000. Fig. 10. Scanning electron micrograph showing the outer surface of the trabeculae (T). The surface is covered with collagenous sheath composed of thick bundles, taking a wavy course (arrowheads). The crosscut surface of the trabecula displays a honeycomb appearance (arrows). x250. Fig. 11. Higher magnification of the outer surface of the trabecula. The collagen bundles take a wavy course, and are enmeshed by a lacework knitted by collagen fibrils (arrowheads). x2,500.

7 Collagen Fibrilar Architecture in the Corpus cavernosum 191 Plate II

8 192 T. Nakano Plate III Fig. 12. Schematic illustration on the architecture of collagen fibrils and smooth muscle fibers in a longitudinally cut surface of the corpus cavernosum. a Flaccid: In the tunica, the collagen bundles take an undulating course, and are arranged in inner circular (C), middle intermingled (I) and outer longitudinal (L) layers. The trabecula (T) ran along the long axis of the corpus, and consisted mainly of bundles of smooth muscle fibers (S). The outer surface of the trabecula is covered with a collagenous sheath composed of thick collagen bundles taking a wavy course (arrows). b Erect (speculation): The collagen bundles in the inner circular (C) middle intermingled (I) and outer longitudinal layers of the tunica appear to be stretched, reducing the thickness of the tunica. The collagen bundles (arrows) covering the smooth muscle fibers (S) in the trabecula (T) seem to be stretched, causing a slenderizing of the trabecula. Asterisk = cavernous space.

9 Collagen Fibrilar Architecture in the Corpus cavernosum 193 Plate III 12a 12b