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Notes: Rabbit articular cartilage was fixed with glutaraldehyde containing Ruthenium Red or Safranin O to localize proteoglycan. By light and electron microscopy, Safranin O proteoglycan localization is easily obtained. Ruthenium Red stained proteoglycan is easily visualized by electron microscopy. Previous digestion with papain prevented staining by either technique confirming that the material was indeed proteoglycan.Using these methods of proteoglycan identification the sites of attachment to collagen are shown as well as the deposition of proteoglycan about cells of differing vitality and the relationship of proteoglycan to cell membranes.

Notes: Granular cell indices (GCI: Dunihue and Robertson, 1957) in kidneys of fetal rats one day after maternal nephrectomy or fetal unilateral nephrectomy on day 21 of gestation were determined by examining sections stained with Bowie's technique. Maternal nephrectomy induced an extreme increase of GCI in fetal kidneys with increased granularity of cells in the juxtaglomerular apparatus. Fetal unilateral nephrectomy did not cause any significant change.

Notes: The sublingual gland of the cat consists primarily of branched secretory tubules that open into an abbreviated duct system. The simple epithelium that composes the secretory tubules consists of an admixture of mucous and serous cells, with the former predominating. Some secretory tubules are capped by a serous demilune. Regardless of position, almost all serous cells have prominent basal folds and border on at least one intercellular canaliculus as well as on the tubule lumen. Serous cells possess an extensive array of irregular, distended cisternae of rough-surfaced endoplasmic reticulum that frequently contain dense intracisternal granules. Serous granules are relatively few in number and rarely show evidence of substructure. Mucous cells, which lack basal folds, contain an apical mass of secretory material in the form of partially fused droplets. The duct system is somewhat less ordered than in most major salivary glands; secretory tubules empty into structures resembling intercalated ducts or may be in direct continuity with ducts intermediate in morphology between intercalated and excretory ducts. The absence of striated ducts noted in this study may be correlated with the high sodium content of cat sublingual saliva. The main excretory duct of the sublingual gland closely resembles that of the cat submandibular gland in terms of morphology, but exhibits little of the transport functions reported in the latter duct.

Notes: In 29 normal persons with complete dental arches, the muscular activity of the temporalis, masseter, medial pterygoid, anterior belly of the digastric, mylohyoid and geniohyoid muscles was studied electromyographically with bipolar fine wire electrodes during various mandibular movements - both resisted and unresisted. Action potentials were recorded on FM magnetic tape and each experiment was also videotaped. Temporalis muscle was active during centric closing of the jaw with either contact of the teeth, or against resistance; during free lateral movements to the ipsilateral side, either against resistance or occlusal contact; during incisor gum chewing, molar gum chewing on ipsilateral or contralateral sides, during normal mastication; and during forceful centric occlusion.Activity occurred in the masseter and medial pterygoid muscles during the following movements: closing the jaw slowly either without occlusal contact or with occlusal contact and against resistance; free lateral movement to contralateral side, either against resistance or with occlusal contact; protraction of the jaw either without occlusal contact or with occlusal contact; swallowing either saliva or water; incisor gum chewing with either the ipsilateral or contralateral molars; normal mastication; and during forceful centric occlusion.Activity occurred in the digastric, mylohyoid and geniohyoid muscles during the following movements; opening of the jaw either slowly or maximally against resistance; closing the jaw against resistance; free lateral movement to ipsilateral and contralateral sides, either against resistance or with occlusal contact; protraction against resistance of the jaw either without or with occlusal contact; swallowing saliva and water; and protraction of the tongue. They work in antagonism (reciprocally) during gum chewing and normal mastication.

Notes: Cell kinetics have been examined in the duodena of intact rats at 6, 16, and 22 days of age, and of hypophysectomized rats at 22 days. In intact rats the crypt population per villus increases more than 10-fold, the greater part of the increase occurring late in the third week. The labelling index does not differ between 6 (28.3%) and 16 (27.6%) days, but increases to 37.8% at 22 days. Generation time also does not differ between 6 (18.0 hours) and 16 (17.7 hours) days, but falls to 10.8 hours at 22 days by shortening of both the presynthetic and synthetic phases. Acceleration of cell migration rate between 18 and19 days of age results in shortening of cell transit time from seven days in rats younger than 18 days to two days in those older than 18 days.When rats are hypophysectomized at six days, the duodenal crypt population per villus at 22 days is comparable to that of intact rats at 16 days. The labelling index at 22 days is 19% below that of intact rats at six days. Generation time at 22 days is slightly shortened by a decrease of the presynthetic phase, but the duration of 15.5 hours is 43% longer than in intact rats at the same age. Despite the small crypt population and the low cell production rate, the cell-transit time in 22-day-old hypophysectomized animals is only three days.

Notes: Earlier investigations of intestinal fat-absorption have stressed the importance of continued protein synthesis to provide membranes which are utilized for the intracellular transport of resynthesized lipid. The resulting membranes, when incorporated into the endoplasmic reticulum (ER) and Golgi complex, serve as vehicles for the movement of fat within the cell and for its release to the extracellular space. In the current study, attention was focused on the morphological changes in the ER and Golgi complex both during fat absorption and at successive time intervals after fat-absorption termination. Morphological interpretations were confirmed by morphometric analysis. This investigation supports the interpretation that during fat absorption, membrane synthesis by the rough endoplasmic reticulum (RER) is insufficient to accomodate membrane utilization and intraconversion, resulting in a decrease of both ER and Golgi complex components. However, following fat-absorption termination, the cell is able to replace previously depleted components of the ER and Golgi complex and regain the full membrane complement of the fasted state. Replenishment of cellular membranes is postulated as resulting from a continued synthesis of new membranes by the RER which eventually exceeds membrane utilized during lipid transport.

Notes: Injections of India ink colored blood, latex, and plastic followed by study of corrosion casts and dissections were used to determine the interconnections of the vertebral venous system and pulmonary circulation in the chicken. Multiple, minute connections are found to the intercostal veins, small mesenteric veins and others connected to the vertebral venous system. Thus, blood can flow in quantities between the vertebral venous system and the pulmonary circulation depending upon pressure gradients.

Notes: The luminal surface ofepithelial cells lining the ductuli efferentes, ductus epididymidis and first portion of the vas deferens were studied with the scanning electron microscope. In ductuli efferentes obvious ciliated cells are identified and it is shown that on non-ciliated cells the short microvilli adhere to one another to form clumps of microvilli. In the epididymis two cell types are described and can be identified as principal cells and clear cells based on the presence or absence of microvilli. Clear cells vary in number along the duct, being most numerous distally. Their surface morphology is also most variable distally. In the vas deferens, principal cells with surface microvilli are easily identified; but in addition, a second cell type is seen, the identity of which is not known.

Notes: Several dissections were performed to determine the level of spinal cord termination and the vertebral level at which the dorsal and ventral roots of spinal nerves C1-S4 emerged from the spinal cord in the rat.These levels of emergence were then compared to the level of exit from the vertebral canal. The dissections demonstrated that the effect of differential growth between spinal cord and vertebral column begins in the lower cervical region and becomes progressively more pronounced throughout thoracic and lumbar levels. The disparity between the vertebral level of emergence of spinal roots from the spinal cord and their level of exit via intervertebral foramina was found to be considerably larger than was previously reported by Greene ('68). It was further noted that the spinal cord terminated at the level of the intervertebral disc between the third and fourth lumbar vertebrae, not between the fourth and fifth lumbar vertebrae as reported by Greene ('68).

Notes: The cytologic development of Meissner corpuscles of the mouse toe pad has been studied using light and electron microscopy, and correlated with silver impregnations of frozen sections. By 18 days of gestation, neurites are seen near the epidermis, but intraepidermal neurites are few. One day after birth, the number of intraepidermal neurites increases, and some accompanying Schwann cells extend their cytoplasmic processes penetrating the basal lamina of the epidermis. Four days after birth, Schwann cells invade the epidermis further, extending many cytoplasmic processes which are intimately associated with basal cells of epidermis. These specialized Schwann cells which contact the epidermis proper also begin to develop cytoplasmic lamellae and thus denote the onset of lamellar cell development. By eight days after birth, the developing lamellar cells become more elaborated, and their cytoplasmic processes contain caveolae and filaments, characteristic features of lamellar cells. This developmental sequence supports the concept that lamellar cells are derived from Schwann cells. Through all stages of development, neurites and Schwann cells interact closely with epidermal cells. Epidermal cells may be essential for corpuscle formation. By 20 to 25 days after birth, mouse toe pad Meissner corpuscles are cytologically mature.

Notes: Developing neuroepithelial cells from the cranial region of neural folds were examined at various intervals from 15 minutes to four hours after administration of teratogenic (250 mg/kg) or embryolethal (500 mg/kg) doses of HU to ICR/DUB mice on ninth day of gestation. Thirty percent of surviving fetuses showed exencephaly after the teratogenic dose. The initial ultrastructural change in some neuroepithelial cells occurred within 15 minutes to one hour after treatment with the teratogenic dose and involved a breakdown of polyribosomes into monosomes which were dispersed evenly in the cytosome. At one to two hours, condensation of the cytoplasm and chromatin was observed along with distorted nuclear shape. At two to four hours, cells exhibited extremely condensed cytoplasm and fragmentation of the cells occurred. The affected cells showed pyknotic nuclei and segregation of fibrillar and granular components of the nucleolus. In some nuclei, particularly after treatment with the higher dose of HU, a condensation of the nucleolus was observed. At this stage images were obtained which indicated that neighboring cells phagocytosed the cellular fragments derived from the HU affected cells.

Notes: Wounds were made in the alveolar process overlying the mesial root of the mandibular first molar of 35-g mice to expose periodontal ligament. Dividing cells were arrested in metaphase by injection of vinblastine sulphate three hours before death three, five and seven days after wounding. Serial 1-m̈m plastic sections of the wound were stained with hematoxylin and eosin. The distance between the mitotic figures and the nearest blood vessel, bone and cementum was measured in every fourth section. No mitotic figure was measured twice. The distances of at least 500 interphase cells from the closest blood vessel were measured in each wound. At all time periods the distribution of mitotic cells was significantly closer to blood vessels than that of interphase cells (p 〈 0.01). From three days to seven days after wounding there was a significant shift of mitotic activity away from the bone side of the ligament, but not away from blood vessels (p 〈 0.05). Approximately 10% of all mitotic cells lay close to cementum, but not close to blood vessels. The majority of the dividing cells were confirmed to be paravascular, and appeared to belong to two populations, one adjacent to bone, the other in the body of the ligament. A third population, not paravascular, lay adjacent to cementum.

Notes: Presecretory ameloblasts were compared morphologically and functionally to secretory ameloblasts in the rat incisor. Structurally, the cell types are similar except for the absence of a Tomes' process at the presecretory stage. The developmental changes at the apical end of the ameloblast were described and correlated sequentially with the onset of extracellular events. First, a layer a amorphous dense material appeared at the dentinal surface adjacent to the ameloblasts. Second, the initial layer of enamel began to develop. Third, inner enamel secretion began with the appearance of interrod enamel. This occurred concomitantly with the appearance of the interdigitating portions of Tomes' processes.Functionally, the protein synthetic activity of presecretory ameloblasts was compared to secretory ameloblasts. Light microscopic radioautography of 1-m̈m thick Epon sections was used to localize 3H-proline and 3H-tyrosine at various times after injection. At time intervals up to 20 minutes the two presursors were localized as a band of labeled protein in the supranuclear cytoplasm of both presecretory and secretory ameloblasts. At 30 minutes an additional band of radioactivity was localized within the apices of both types of ameloblasts. In presecretory cells the apical reaction band was over the proximal portion of Tomes' processes which border on the dentin. In the secretory cells, the apical reaction band was over both proximal and interdigitating portions of Tomes' processes and over the enamel. Grain counts over the secretory ameloblasts showed that the incorporation of tyrosine increased by 7.5% as opposed to a 63% increase with proline when compared to the values in presecretory cells. The different increases with the two precursors were in keeping with the different amounts of the two amino acids reported to be present in enamel protein.It was concluded that while the secretory ameloblasts synthesize and secrete enamel protein, both presecretory and secretory cell types produce another category of protein which is involved in the apical reaction band. It was proposed that this material is structural protein being contributed to the continuously lengthening Tomes' process which is speculated to occur during formation of enamel.

Notes: Tritiated-L-proline was injected into the right Gasserian ganglion of mature rats for incorporation into proteins being carried by rapid axonal transport to nerve endings. The distribution of the nerve endings in molar gingivae and periodontium was subsequently mapped in serial sections by autoradiography. We found that sensory nerve terminals are most prominent in two regions of the free gingiva: (1) In the junctional epithelium there is a rich innervation of the basal epithelial layer with many endings penetrating several cell layers. (2) In the crestal epithelium arborized endings are present, especially on the marginal side of the crest. In the attached gingiva a few terminals are found in the epithelium with more in the lamina propria; the latter appear to be encapsulated. The periodontal ligament is only moderately labeled with silver grains located over nerve bundles, over nerves associated with blood vessels, and over a few apparent endings in the apical region. In the cementum no labeled endings are seen. Labeling of Gasserian cell bodies primarily reveals specific intra-epithelial nerve endings in gingival crest and junctional epithelium. The absence of major labeling of the periodontal ligament supports the view that many nerves in that region are derived from cell bodies in the mesencephalic nucleus.

Notes: Within 45 minutes after mating in the rabbit, numerous heterophil leukocytes adhere to the endothelium of venules in the vagina. Initial association appears to occur via small protuberances from the leukocyte which fit into small indentions in the endothelial cell. Following adherence, leukocytes flatten and pass between endothelial cells. A regular intercellular space separates the leukocyte from the endothelial cells. Leukocytes subsequently migrate through the connective tissue to the epithelium. By three hours postcoitus, the region beneath the basement lamina of the vaginal epithelium is crowded with numerous juxtaposed leukocytes. Leukocytes subsequently migrate between epithelial cells into the vaginal lumen where they actively engulf spermatozoa. Spermatozoa appear to be ingested head first. Numerous small filaments are observed in the leukocyte cytoplasm in the region adjacent to the sperm head. Degranulation of azurophyl granules follows sperm uptake. The leu kocyte response can be elicited either by spermatozoa (from the epididymis) or by semen (from vasectomized bucks), but is not elicited by ovulation (with human chorionic gonadotropin). It is suggested that the response may be initiated because the vagina does not distinguish between semen, spermatozoa and bacterial infection.

Notes: Twenty-four hours after mating in the rabbit, numerous spermatozoa are observed attached to the surface of the vaginal epithelium. Spermatozoa appear to be attached by their heads to microvilli of the simple columnar cells which compose the vaginal epithelium. Spermatozoa are taken up by the epithelial cells, and they are found within the cells in various stages of de generation up to seven days after mating. Epithelial cells frequently appear to be filled with numerous vacuoles containing sperm components in various stages of degeneration. It is suggested that some property of the surface of sperm heads may render them particularly susceptible to phagocytosis. Phagocytosis of spermatozoa may not have long-term harmful effects for the epithelium since these epithelial cells presumably have a high rate of turnover.

Notes: Nonjunctional and nonsynaptic membranes of hair cells in the chinchilla organ of Corti were examined using the freeze-fracture technique. Cy-toplasmic leaflets of the apical membranes of hair cells have particles, 6-12 nm in diameter, but many more particles are found on apical membranes of outer hair cells than on inner hair cells. Cytoplasmic leaflets of the lateral membranes of outer hair cells are covered with large particles, but the corresponding regions of the inner hair cell membrane have fewer particles and these are small or medium-sized. Two types of particle aggregate also distinguish this region of the inner hair cell. The first consists of patches of particles closely spaced in rec tilinear arrays. The second consists of parallel strands of widely spaced large particles similar in size, but not in distribution, to the large particles on the lateral membranes of the outer hair cells. The basal membrane of outer hair cells is distinguished from that of inner hair cells by plaques consisting of cross-hatched in-cisures in the external membrane leaflet. While the significance of these anatomical features is not yet apparent, they give the hair cell plasmalemma a richness of regional specialization found in few other cells and suggest that there are important functional differences between inner and outer hair cells.

Notes: A scanning electron microscopic (SEM) study of palatal mucosa maintained in organ culture (50% O2-Eagle's MEM w/o serum) from 12 to 360 hours is presented. The morphological changes occurring in the explants during culture are illustrated and the findings are compared with those observed using transmission electron microscopy and light microscopy. The results confirm that cellular fine structure in epithelium and connective tissue is more easily visualized when SEM is employed following in vitro maintenance of explants as opposed to examination of fresh uncultured biopsies. The in vitro system used would appear to be applicable to studies designed to investigate the effect of various substances on cell proliferation and cell interactions.

Notes: Pulp development was studied with light and electron microscopy in apical portions of cat primary and permanent incisors at various stages of development in order to qualitatively assess hypothesized differences during maturation. Primary and permanent tooth pulps passed through similar stages up to when the primary teeth began to resorb. At that point permanent tooth pulps continued to develop in complexity relative to myelinated axons and developed thin epineurial sheaths. Although the stages were similar in several regards, the time needed by the permanent tooth pulps to reach each stage was considerably greater than for the primary tooth pulps. It is of interest that the young permanent teeth had only a small portion of their final complement of nerves in spite of their relatively complete development.Individual axons, especially myelinated axons, appeared more immature in younger primary and permanent teeth. This suggests a less mature sensory capability for erupting primary teeth as compared to fully developed primary teeth as well as for erupting and young permanent teeth compared to old permanent teeth.

Notes: Nerve endings were observed between collagen fiber bundles in the alveolar compartment of the periodontal ligament. In close relation to these endings, cells were observed with a rounded cell body from which cytoplasmic extensions protruded, each forming part of a sheath around the terminal region of a small nerve fiber. The nuclei of these cells occupied an eccentric position in the cytoplasm and were characterized by a typical kidney shape. In the cell's center a prominent Golgi complex was present. In the peripheral cytoplasm ladder-like structures with a periodicity of 160 nm were occasionally observed. The cell bodies were surrounded by a basal lamina, while the cytoplasmic extensions investing the nerve endings were surrounded by an envelope consisting of alternating electron dense and electron translucent layers containing material that stained with PAS and Alcian Blue. Where these envelopes bordered on the connective tissue, areas of filamentous material with a periodicity of 100-150 nm were occasionally found. The end organs may be considered to form part of a receptor system.

Notes: Contact sites between lymphocytes and between lymphocytes and macrophages were demonstrated by electron microscopy in the lymphoid follicles of the bursa of Fabricius. When compared with nonspecialized regions of the cell membranes, these contact sites were characterized by a decreased intercellular distance, subplasmalemmal densities and coated pits. Microfilaments, microtubules and coated vesicles of the subjacent cytoplasm were frequently associated with these contact sites. When the same cells were isolated and introduced into culture, they formed cluster-like assemblies in which cells were closely approximated along broadly contacting surfaces. The morphology of the sites appeared to involve primarily the plasma membrane (including coated pits) and the cell coat. These observations indicated that the same cells of a given lymphoid tissue can form one type of contact site in vivo and another, dissimilar type of contact site, in vitro.

Notes: The spleens of rats were studied by light and electron microscopy during the course of an acute myelogenous leukemia, with special reference to infiltration of leukemic myeloblasts in the spleens and to the correlation of leukemic cell infiltration with splenic hematopoiesis and splenomegaly. Leukemic myelo-blasts infiltrated the cordal space of the red pulp. Many of them appeared in groups. Even in spleens which were heavily infiltrated, leukemic myeloblasts did not penetrate the white pulp. Massive infiltration and proliferation of the leukemic myeloblasts in the red pulp resulted in splenomegaly. The spleen increased its he-matopoietic activity, while the medullary hematopoiesis diminished due to the invasion of leukemic myeloblasts in the bone marrow. Compensatory splenic hemato-poiesis occurred in most of the leukemic spleens, but diminished in spleens which were very heavily infiltrated with leukemic myeloblasts. Thus, the degree of splenomegaly and splenic hematopoiesis did not necessarily correspond to the percentage of leukemic myeloblasts in the bone marrow, but rather related to the number of leukemic myeloblasts present in the spleen. A possible role for the splenic sinus walls in promoting compensatory hematopoiesis in the spleen is discussed. A consistent association of type “C” virus particles with leukemic myeloblasts was observed.

Notes: We describe morphological sex differences in urinary bladders of the urodele amphibian Necturus maculosus. The mucosal epithelial cells of bladders from males were tall and contained considerable PAS-positive material. In scanning electron micrographs of the mucosal surface, epithelial cells from male bladders were well demarcated and were capped with microplicae or with long cilia. The mucosal epithelial cells of bladders from females were low and contained only a small amount of PAS-positive material; in scanning electron micrographs cell boundaries could not be distinguished and no cilia or microplicae were present. Bladders from males had higher transepithelial potential difference and lower water content than bladders from females. Urine analyses were not significantly different in the two sexes. It is suggested that response to androgens in the male accounts for the observed differences.

Notes: During renewal of the enamel organ in the rat incisor cohorts of epithelial cells are transported sequentially through presecretory, secretory and maturation zones to the gingival margin where the life cycles of these cells terminate. This process was examined kinetically by determining the absolute flux of cells within each of these zones of amelogenesis. It was found that the efflux of ameloblasts, stratum intermedium and papillary layer cells from the presecretory zone was about equal to the efflux plus expected growth within the secretory zone. However, between the secretory and maturation zones about 50% more ameloblasts entered the maturation zone than were required to account for the egress at the gingival margin and the expected growth. Since there was no similar imbalance between these zones for papillary layer cells, it was concluded that this discrepancy must represent a 50% reduction in the size of the ameloblast population during the maturation stage of amelogenesis. It was calculated that a little over 25% of the loss occurred immediately at the start of maturation within the region of postsecretory transition and the remaining 25% of the loss occurred throughout the subsequent regions of the maturation zone. In addition to the kinetic analysis graphic reconstructions, or surface maps, of ameloblast nuclei were prepared. These maps illustrated the characteristics of ameloblast nuclear packing within the three zones of amelogenesis and they provided quantitative confirmation that as ameloblasts progress through the maturation zone, there is a loss of cells in an amount predicted by the kinetic analysis.

Notes: Presence of a ridge of fatty tissue running obliquely upwards and backwards over the left aspect of the pulmonary trunk was observed. It started from the left end of the infundibulo-truncal junction and after running parallel adjacent to free anterior border of the left auricle, terminated into an expanded, oval and discoid structure, a little above the level of the tip of the left auricle. The structure was found to be of constant occurrence, position and form.A comparison between similar structure reported in relation with the aorta and the right auricle was attempted.

Notes: The morphology of the thyroid gland of the woodchuck, Marmota monax, was studied during the four seasons of the year. In the spring the thyroid is extremely heterogeneous in appearance. Some follicular cells appear quite active. They contain a well defined Golgi apparatus, abundant large colloid droplets and pseudopodia but few, if any, apical vesicles. Other less active cells have poorly defined rough surfaced endoplasmic reticulum and lack a well developed Golgi apparatus. They do not contain apical vesicles or colloid droplets. Summer thyroids have uniformly small follicles which are lined by high cuboidal cells containing numerous mitochondria, apical vesicles, abundant rough surfaced endoplasmic reticulum, and lipid droplets but few colloid droplets. There is extensive lateral and basal infolding of the cytoplasmic membranes in these cells. In the fall and winter the follicles are larger than in the summer and contain more colloid. Numerous heterogeneous dense bodies appear in the cytoplasm of the follicular cells in the fall and increase in number in the winter when there is an obvious sparsity of such glycoprotein synthetic organelles as Golgi apparatus and rough surfaced endoplasmic reticulum. These morphologic changes are compared with previous studies of thyroid structure and function in other animals and are correlated with the seasonal physiologic activities of the woodchuck.

Notes: The radioactivity decay of the mitochondrial compartment from the zona fasciculata of the adrenal cortex of 3H-thymidine-injected rats was followed by high resolution autoradiography. The number of days in which the radioactivity of the mitochondrial compartment was reduced to a half was calculated from the semilogarithmic plots of radioactivity versus time. Since DNA is a very stable molecule, it was assumed that this parameter can be an estimate of the half-life of adrenocortical mitochondria. The half-life of mitochondria from the zona fasciculata of the normal rat averaged 11.17 days, and ACTH was found to increase significantly this figure to about 15 days. It is hypothesized that the ACTH-elicited stimulation of the growth of rat adrenal zona fasciculata mitochondria involves not only hypertrophy and proliferation of the organelles (Nussdorfer et al., '74b), but also the slowing down of the degeneration rate of mitochondria.

Notes: Responses of the lateral motor column (LMC) in the frog spinal cord to various experimental manipulations generally are manifested in an adjustment of the number and sizes of the motor neurons. Analysis of the brachial LMC in induced-triploid Rana pipiens larvae as compared to diploid controls revealed a significant increase in nuclear size with a concomitant reduction in cell counts at all stages of development examined. These compensatory responses of the LMC to polyploidy in normal size frog larvae are consistent with results reported for other amphibian polyploid tissues and serve as a basis for exploring mechanisms of plasticity in the developing central nervous system.

Notes: The degenerative and early regenerative events following mincing and autotransplantation of rat skeletal muscle were examined at the ultrastructural level. During the first eight hours after injury, myonuclei undergo pyknosis, mitochondria become enlarged and vesiculated, myofilaments appear less distinct than normal and the sarcolemmata either disappear or become extensively fragmented. Further degeneration of the myofibers progresses slowly until macrophages and polymorphonuclear neutrophils invade the degenerating sarcoplasm between one to four days after mincing. Scattered throughout the minced muscle implant during the first 24 hours after injury are small, viable-appearing, undifferentiated cells located between the external lamina and degenerating sarcoplasm. Such cells, which are structurally similar to satellite cells seen in uninjured muscle, are believed to be regenerating presumptive myoblasts due to their mesenchymal-like morphology and sublaminar position. External laminae of the injured muscle fibers do not undergo immediate degenerative changes, but rather persist during the first three to six days as laminar tubes within which spindle-shaped myoblasts and newly formed myotubes are frequently observed. Examples of regenerating myoblasts in the process of budding-off from damaged muscle fibers were not observed in this study. Therefore, the evidence suggests that satellite cells are the major source of regenerating myoblasts in skeletal muscle of the rat.

Notes: Autografts of tongue onto the liver contained taste buds without nerves for up to 30 days. However, these denervated taste buds were generally smaller than normal and distorted in structure. Nerve fibers invaded the graft from the liver and after 30 days normal appearing taste buds were found only in implants of tongue which contained extensive nerve fibers. Thus it appears that certain visceral nerves can maintain and possibly induce taste bud formation in implants of tongue. This is interpreted as additional evidence for the trophic influence of nerves upon taste buds. Moreover, this ability to maintain taste buds is not restricted to gustatory nerves in the newt.

Notes: In an attempt to facilitate quantitative analysis of neural peri-karya, an inexpensive system of gridded coverslips has been developed to permanently map sections of tissue on microscopic slides. Utilizing common photographic copying techniques with Kodalith film, a mylar coverslip is produced which is imprinted with a grid of known dimensions. The exact scale of the grid can be varied precisely for compatibility with a particular field of magnification. The versatility and economy of this technique make it useful in many microscopic studies requiring superimposition of discrete landmarks.

Notes: A microanatomical study of the immature rat calvarium was performed in order to develop a method for the isolation of osteoblasts by enzymatic means. Although generalized osteogenesis was evident in fetal rats, differential growth patterns were observed beginning at 19 days in utero. Considerable portions of the endocranial periosteal bone surface were lined by flattened, less active cells; discrete areas also contained multinucleated osteoclasts. Cell counts of whole calvaria revealed that one fifth of the total cell population were osteoblasts, most of which were located in the central portions of the frontal and parietal bones. Prior excision of these segments permitted the subsequent removal of virtually all periosteal tissues. Cleaned 19-day fetal bones, incubated in crude collagenase for two hours, released about 40,000 cells/calvarium, consisting of 85-90% osteoblasts and lesser amounts of connective tissue and bone marrow elements. Because of the relatively small sizes of most extraneous cells, purity on a cell volume basis was approximately 95%. It is predicted this preparation will be useful in the investigation of certain aspects of osteoblastic function.

Notes: A reliable and uniform vascular perfusion fixation method for the testis has been developed by using an initial washout solution containing a vasodilator and an anticoagulant. This is followed by a brief fixation with a sodium phosphate buffered formaldehyde-glutaraldehyde solution of conventional strength, and then a second more concentrated aldehyde fixative solution containing picric acid. The method takes into account some of the unique features of the vascular supply of the male genital tract for its favorable perfusion and fixation. The advantages of this method are: (1) consistently favorable preservation of the testis; (2) simple and inexpensive apparatus; and (3) stable and relatively innocuous stock solutions.

Notes: The time required for ovarian follicular development in the cyclic hamster was determined by marking follicles with [3H]-thymidine on day 1 of the estrous cycle (day of ovulation) and by following their fate over the next three cycles by autoradiography. A model of follicular development was formulated, based on the total number of follicles and the changing percentage of labelled follicles with time.It was estimated that 20 days elapsed before a preantral follicle with two to three layers of granulosa cells (stage 1) was able to ovulate. Follicles spent eight days in stage 1 and an additional eight days in stage 2 (4-5 granulosa layers). Preantral follicles entering stage 3 (6-7 granulosa layers) and 4 ( 8 layers) on day 1 of the cycle ovulated four days later.Approximately 30 follicles per ovary entered and left stage 1 each cycle, representing a 50% turnover. Of the follicles leaving stage 1, about 50% were transformed into stage 2 follicles; the remaining ones underwent atresia. Stage 2 follicles also had a 50% turnover each cycle. Half of the stage 2 follicles underwent atresia; the remainder developed into stages 3 and 4 follicles Fifty percent of the stages 3 and 4 follicles present on day 1 of the cycle ovulated at the next estrus with the remainder undergoing atresia.

Notes: Half of the rabbits developed antisperm antibodies (measured by either indirect immunofluorescence or sperm immobilization tests) after either a unilateral or bilateral vasectomy. The raised antibody levels, particularly six months or longer after vasectomy, often accompanied patchy orchitis. Seminiferous tubules from such animals exhibited sloughed, multinucleated, and immature germinal cells which were engulfed by phagocytic cells. Mononuclear infiltrates were occasionally present. The basal lamina infolded and thickened by means of supernumerary layers and appeared to be endocytosed by cells of the seminiferous tubules. Four months after vasectomy, numerous phagocytic cells were seen to migrate through the intact epithelium of zone 1 in the caput epididymidis, and were particularly prevalent in animals that exhibited testicular damage. These macrophages may serve to present sperm antigens to lymphocytes.

Notes: The embryogenesis of the proctodeal gland and development of the connective tissue of the associated lamina propria in the dorsal wall of the proctodeum of Common Coturnix (Coturnix c. japonica) were studied on embryos collected at 12-hour intervals from day 7 of incubation through hatching. Gland development began at 9.5 days from ectodermal-derived epithelial buds developing in the craniolateral region of the dorsal proctodeal epithelium. At 11 days, the epithelial buds had become solid convoluted epithelial cords which, by 11.5 days branched to form solid individual glandular units. Lumina of the unit were formed by degeneration and vacuolization of the central cells of these solid units. Squamous epithelial caps, several layers thick, separated the vacuolizing units from the proctodeal cavity at day 13; these caps subsequently thinned, bulged into the proctodeal cavity and, by day 16, began to disappear opening the lumen of the units to the proctodeal cavity.Sequential formation of epithelial buds, by region, was: craniolateral at 9.5 days, craniomedial expansion at 10.5 days followed, beginning at 11 days, by cau-dal expansion along entire dorsal proctodeal wall. Sequence of appearance of gland-associated connective tissue fibers was: 9.5 days, reticular fibers already present; 11 days, collagen fibers; 11.5 days elastic fibers.Serially reconstructed models of an individual glandular unit at hatching enabled classification of the unit as a simple branched alveolar gland.

Notes: An adenylate cyclase highly responsive to stimulation by parathyroid hormone (PTH) and calcitonin (CT) in vitro was observed at certain times during normal prenatal development of the maxillary-palatal process complex in the golden hamster. Responses of the enzyme to these hormones were barely detectible at the earliest stage examined (day 10/20). The enzyme became extremely sensitive to activation by either hormone during the time of rapid growth of the palatal processes (day 11/20) and during fusion between the palatal processes (day 12/20). Thereafter, responses were greatly diminished and little or no activation of adenylate cyclase was observed until birth. Adenylate cyclase from fetuses in which clefts of the secondary palate were induced by maternal treatment with hydrocortisone (50 mg) on day 11/3 also displayed an enhanced sensitivity to PTH and CT on day 11/20, but the sensitivity of the enzyme was greatly decreased from that in normal animals during the normal time of palatal fusion (day 12/20) and was barely detectible or absent at the remaining time periods studied (days 13/20 and 14/20). Addition of hydrocortisone to the incubation mixture, either separately or in combination with PTH or CT, did not remarkably affect the response of adenylate cyclase to these hormones. Moreover, the appearance of the adenylate cyclase sensitive to hormonal activation did not result from changes in phosphodiesterase activity during palatal maturation.

Notes: Human spermatozoa display unusually limited affinities in their interaction with oocytes of other species. They adhered to and, when capacitated, penetrated the vestments of the oocyte of an ape-the gibbon, Hylobates lar- both in vivo and in vitro. On the other hand, human spermatozoa would not even attach to the zona surface of sub-hominoid primate (baboon, rhesus monkey, squirrel monkey), nor to the non-primate eutherian oocytes tested. Among the apes the gibbon stands furthest from man. Thus, although the specificity of human spermatozoa is not confined to man alone, it probably is restricted to the Hominoidea. This study also suggests that the evolution of man and perhaps the other hominids has been accompanied by a restrictive change in the nature of the sperm surface which has limited and made more specific the complementary surface to which their spermatozoa may adhere. For the failure of human spermatozoa to attach to the zona surface of all non-hominoid oocytes stands in contrast to the behaviour of spermatozoa of the several other mammals studied which, in most combinations, adhered readily to foreign oocytes, including those of man. Taxonomically, the demonstration of a compatibility between the gametes of man and gibbon, not shared with cercopithecids, constitutes further evidence for inclusion of the Hylobatidae within the Hominoidea.

Notes: A histological, ultrastructural and histochemical study of the mandibular gland of the Australian possum Trichosurus vulpecula revealed essentially similar features as those described earlier for the mandibular gland of the taxonomically relatively unrelated American opossum Didelphis. The secretory endpieces consist of a branched tubular part, composed of serous cells whose secretion granules contain neutral glycoproteins, and terminal acini, consisting of seromucous cells containing small amounts of sialomucins. Relatively short intercalated ducts lead to striated ducts of variable ultrastructural appearance. The striated ducts run in bundles in the center of each sublobule of the gland. The possible functional significance of the abundance and variability in ultrastructure of the striated ducts is discussed.

Notes: Inflammatory exudate (SE) cells were collected from subcutaneous coverslips in mice and transferred into lethally irradiated (1,000 r) recipients. Eight days after transplantation 59Fe incorporation in the spleen and bone marrow was significantly greater than in controls treated with the suspending medium only. One hundred percent of mitoses were of the T6T6 karyotype in the marrow and spleen when SE cells were obtained from CBA/T6T6 donors. The repopulating potential of SE cells, however, lagged significantly, behind that of bone marrow cells and the failure to observe consistently macroscopic spleen colonies calls into question whether the observed regeneration was due to pluripo-tent stem cells. Radioautographic studies with 3H-TdR showed that the majority of SE cells had recently been generated, but long-lived, noncycling cells of lymphoid and monocytoid morphology were also present in the exudate.

Notes: The ultrastructure or sarcoplasmic reticulum (SR) was studied in the conducting fibers of the dog heart. A dense network of sarcoplasmic tubules occurred in well preserved cytoplasmic areas. Some of the tubules extended into large sacs filled with finely filamentous material. The sacs often appeared in subsarcolemmal apposition connected to the sarcolemma by electron dense projections. The flattened cisternae of SR occurred beneath the sarcolem-ma in the myofibrillar region of the cell. Long flattened cisternae only partially apposed to the sarcolemma were quite common in one specimen. Sarcoplasmic reticulum was continuous throughout the interfibrillar spaces surrounding each myofibril. At the level of the Z-line the SR formed a quasi tubular flattened structure surrounding the Z-line and closely adhering to it.

Notes: The object of the present study was to examine ovarian oocytes of the sciuromorph rodent. Thomomys townsendii, to determine if they contained cytoplasmic inclusions similar to those reported in myomorph rodents. Our observations indicated the presence of cytoplasmic lamellar inclusions of oocytes of primary, secondary and large vesicular follicles of Thomomys. Small primary oocytes contained numerous cytoplasmic lamellae which appeared as spirals or concentric rings in cross-section. Longitudinal sections suggested the inclusions were arranged as concentric cylindrical sheets of material. Secondary and vesicular oocytes had morphologically similar inclusions, but, in addition, often had a tubular element of the endoplasmic reticulum associated with the core of the lamellae. Tangential sections through the lamellae revealed a crystalline substructure, regardless of the age of the oocyte. Our results indicate that cytoplasmic lamellar inclusions are not restricted to myomorph rodents, but may well be present in other species. The lamellar inclusions are three-dimensionally most similar in structure to those described in the hamster, but several fine structural differences exist even between these species. Thomomys is the only species to date in which a close topographical association develops between the lamellar inclusions and elements of the endoplasmic reticulum. Since the lamellae-endoplasmic reticulum association was not observed in the youngest oocytes examined, it is unlikely the endoplasmic reticulum is involved in synthesis of the lamellae, but it may, nonetheless, be metabolically related to the lamellae.

Notes: The dorsal motor nucleus of the vagus nerve (DMX) of adult cats and young kittens was studied by quantitative light microscopic methods. In normal animals, the DMX was found to contain no distinct subgroupings of neurons, based on somatic volume or Nissl pattern. Retrograde perikaryal responses to axotomy of neurons in the DMX were found to be of a more subtle nature than those seen in other types of neurons. Quantitative methodology applied to the DMX after cervical vagotomy permitted a better understanding of the results of axotomy than could be obtained by routine microscopic observations. Changes which occurred included a slight chromatolytic reaction, and a decrease in the volume of the nucleus followed by an increase in somatic volume. These morphological alterations were affected by the factors of age of the animal, time after axotomy, and length of the intact proximal axon stump. More pronounced perikaryal changes occurred when the vagus nerve was recut at a more proximal level five days after the first vagotomy. Interpretation of the data yielded the conclusion that most if not all neurons of the ipsilateral DMX contribute axons to the cervical vagus nerge. In addition, at least 10% of the neurons on the side contralateral to vagotomy showed signs of retrograde reaction. It was therefore concluded that there exists in the vagus nerve a population of axons with cell bodies located in the contralateral DMX.

Notes: Freeze-fracture combined with quantitative electron microscopy of the intact human erythrocyte (RBC) and ghost revealed significant differences in their intramembranous particle coefficients. External (E) fracture-faces of unfixed ghost membranes were found to contain 40% fewer particles than those of intact unfixed RBC. The particle distribution of the intact RBC membrane depended on the use of glutaraldehyde fixation and glycerol cryoprotection. Whereas glutaraldehyde- and glycerol-treated cells disclosed 70% fewer E-face particles than did intact unfixed cells, poly-L-lysine-treated, intact, unfixed RBC showed no such differences. Treatment with a combination of poly-L-lysine and glutaraldehyde, however, increased the amount of E-face particles while reducing those of the protoplasmic (P) face. The poly-L-lysine effect varied with its concentration and was unaffected by previous application of neuraminidase. Nor did the lectin phytohemagglutinin induce particle rearrangement in intact cells.Our data demonstrate that the processes of glutaraldehyde fixation and glycerol cryoprotection modify the RBC membrane by decreasing the number of E-face particles present. In addition, the combination of poly-L-lysine and glutaral-dehyde alters the affinity of some particles for one half of the membrane, suggesting that in freeze-fractured RBC, chemical bonds formed at the extracellular surface of the membrane can influence particle partitioning.

Notes: Tight junctions (zonulae occludentes) create a pericellular barrier to the diffusion of large molecules in non-keratinizing mammalian epithelia. However, in cornifying epithelia such as the epidermis, the importance of tight-junctional elements versus secreted intercellular lipid for barrier function is uncertain. In an attempt to resolve this question, we compared membrane structure in the stratum granulosum and stratum corneum of epidermis, esophagus, and vagina of newborn and adult humans and mice under both normal and various experimental conditions. We incubated pieces of epidermis in organ culture and infused tissues with lanthanum or horseradish peroxidase in vivo and in vitro. All were processed for electron microscopy of freeze-fracture replicas or thin sections. Lanthanum seeped outward to the stratum granulosum in all tissues examined-further apical migration was halted by lamellar-body contents in skin. A similar pattern of intercellular lamellar lipid deposition and membrane structure occurred in all epithelia studied. Freeze-fracture replicas of these obstructive regions revealed occasional, incomplete junctional strands (particularly in moist epithelia) and abundant lamellar material, but complete zonulae occludentes were never encountered. A possible relationship between moisture and tight junction formation was further suggested by organ culture experiments during which brief incubations stimulated an increase in the number of junctional strands and diminished numbers of lamellar bodies. We conclude that, in the epithelia studied, the deposition of secreted lamellar body contents forms the barrier to water-soluble tracer loss: tight-junctional elements are either absent or too fragmentary to constitute an effective barrier.

Notes: The distribution of axons in the posterior articular nerves in rhesus monkey has been studied in terms of ratio of myelinated to unmyelinated fibers, and size of myelinated fibers. Conduction velocity measurements were also made. The nerves contained 2,000 to 2,200 unmyelinated fibers of 0.2 to 1.2 μm. diameter. They contained 399 to 478 myelinated fibers, varying in size from 1.4 to 12.3 μm. Thus 80 to 85% of fibers in these nerves were unmyelinated. The maximum conduction velocity of 70 to 80 meters per second corresponded to an initial small deflection, with most fibers conducting at slower rates. These results are compared to previous reports which severely underestimated the number of unmyelinated fibers because electron microscope counts were not utilized.It is suggested that pain fibers from the knee joint of monkey make up much of this large population of unmyelinated axons. An unknown number of post-ganglionic sympathetic fibers is also included in the unmyelinated fiber group.

Notes: In this article a description of the process of spermatogenesis in the Chinese hamster is given.Spermiogenesis could be divided into 16 steps. The cycle of the seminiferous epithelium was divided into 12 stages, coinciding with the first 12 steps of spermiogenesis. The relative and absolute duration of the stages was determined. The duration of the cycle of the seminiferous epithelium was found to be 17.0 days.The morphology of the spermatogonia was studied in seminiferous tubules mounted “in toto.” Four classes of spermatogonia could be discerned: undifferentiated A spermatogonia (Ais, Apr, Aal), differentiated A spermatogonia (A1, A2, A3). In spermatogonia and B spermatogonia (B1, B2). It is interesting to note that the last generation of spermatogonia (B2) arises at the beginning of stage 7 and divides to give rise to primary spermatocytes in the second half of this stage; in most other species the last generation of spermatogonia arises in stage 4, giving rise to primary spermatocytes in stage 6.The undifferentiated A spermatogonia were counted in six stages of the cycle, together with the differentiated A, In or B spermatogonia present in the same stages. The results of these cell counts are discussed in detail. One of the conclusions that could be drawn about the behaviour of the Ais and Apr spermatogonia during the cycle of the epithelium was that there is mitotic activity in these cells in several stages of the cycle. It is suggested that this mitotic activity serves to generate the Aal spermatogonia, which after one or more divisions transform into A1 spermatogonia between stages 2 and 8.

Notes: Incubation of early embryos of Nothobranchius korthausae in sterile Tris-buffered pronase with added salts and EDTA results in complete dechorionation, after which development proceeds normally and at the same rate as in control embryos. The time required for dechorionation varies according to the age of the embryo and the concentration of pronase employed.

Notes: Although a number of morphological studies have investigated the vascular system of the rat kidney, minimal data are available on the detailed anatomy of the efferent arterioles located throughout the cortex of the kidney. In the present study, the renal vascular system was filled with Microfil and the various efferent arteriole patterns were examined extensively. The efferent vessels of the entire cortex appear to form three major patterns which in turn divided the cortex into three separate regions: the outer, middle and inner cortex. The efferent arterioles of the outer cortex leave the glomerulus and run perpendicular to the kidney capsule. However, as the efferent arterioles ascend, they may show three variations in the way they branch: (1a) the efferent arteriole does not branch until directly beneath the capsule, (1b) the efferent vessel begins to divide into its major branches 100-200 μm below the surface of the kidney and (1c) the efferent vessel has only a short course before giving off many side branches. In the middle cortical area, the branches of the efferent arteriole run lateral to the glomerulus. However, the efferent arterioles of the inner cortex have a few branches which run lateral to the glomerulus while most of them descend into the medulla as vasa rectae. The unique morphological features of the efferent arterioles of the outer cortex are of particular interest in light of the functional data which suggests that the reabsorption of fluid by peritubular capillaries may indeed regulate the rate of net tubular sodium reabsorption.

Notes: The differentiation of leukocytes in the bone marrow and blood of normal adult male rats was studied by electron microscopy and peroxidase cytochemistry. Tissue samples were fixed in glutaraldehyde, or paraformaldehyde-glutaraldehyde, and incubated in a peroxidase medium containing 3,3′-diaminobenzidine and H2O2 at pH 7.6. Mature cells of blood were identified, and then the earlier stages of maturation in bone marrow were analyzed. In immature cells of four cell lines, neutrophils, monocytes, basophils, and eosinophils, peroxidase is synthesized and could be demonstrated in the rough endoplasmic reticulum (RER), Golgi complex, and in cytoplasmic granules. Later in maturation, reaction product for peroxidase could not be found in RER or Golgi complex, indicating that peroxidase synthesis had ceased. In two cell lines, neutrophils and monocytes, peroxidase-negative granules were formed, and the mature cells contained two populations of cytochemically distinct granules. All granules of mature eosinophils were peroxidasepositive. In mature basophils, some granules were clearly peroxidase-positive; others displayed variable density, making interpretation uncertain. Mast cells were never seen in blood, but were abundant in bone marrow; peroxidase was never found in their granules by either electron microscopic cytochemistry or a variety of light microscopic methods. Hence, these cells differ from basophils, not only in morphology but also in the enzyme content of their granules.

Notes: An experiment designed to study the effects of the copper IUD on the virgin rat uterus has revealed the presence of intracellular collagen fibrils in control uteri and in uteri that have contained a copper IUD for three months. The cells containing the collagen are found in the stroma in close proximity to the uterine epithelium. The collagen is found within membrane-bound cytoplasmic vacuoles that vary in morphology. In some cases the fibrils are tightly packed and linear, with no other material evident in the vacuole. In other examples, the fibrils are randomly arranged and the vacuoles contain a punctate material which is characteristic of phagolysosomes. Finally, cytoplasmic vacuoles are seen which contain ill-defined debris and poorly-visualized structures that exhibit a periodicity, suggesting a terminal phase of fibril breakdown. All animals were sacrificed in metestrus, and the results indicate that intracellular collagen is present in the nulliparous rat uterus at this stage of the cycle. In addition, this phenomenon does not appear to be influenced by the presence of a copper IUD over a period of three months.

Notes: Electronmicrographs of both the vermis and hemisphere of the cerebellum of the squirrel monkey (Saimiri sciureus) show numerous granule cell bodies partially or completely surrounded by myelin. The myelin is of the compact type and consists of 1 to 13 lamellae. In several cases of partially ensheathed cells the myelin is clearly derived from extensions of myelin sheaths that surround small caliber axons. Either all or only the outer lamellae surrounding the axon contribute to the extensions. In the first instance the myelin buckles at its mesaxon pole and the resulting doubled flap extends for a variable distance along the cell surface.

Notes: Pieces of upper lip and lower jaw from 13-day old mouse embryos grown in organ culture with excess vitamin A showed metaplastic changes in epidermis and hair follicles after 7 to 21 days. Changes were less marked in upper lip explants from 15-day old embryos. Areas of keratinizing epidermis showed a much higher incidence of Alcian blue-positive bodies when excess vitamin A was present. Histochemical tests with Alcian blue at critical electrolyte concentrations indicated moderately sulfated acidic mucosubstances in these bodies. Other patches of epidermis were transformed into stratified cuboidal epithelium producing PAS-positive. Alcian blue-negative, mucosubstances. The hair follicles, transformed by vitamin A into glands, developed three types of epithelium with apical granules, luminal borders and occasional goblet cells, all showing mucosubstances. Tests suggest that both metaplastic epidermis and glands secrete either neutral mucosubstances or non-sulfated acidic mucosubstances. Another response to vitamin A was a rapid loss of glycogen, particularly in hair follicles and the epidermal basal layer. It was concluded that excess vitamin A alters not only the morphogenesis but also the distribution and synthesis of polysaccharides in developing skin.

Notes: Small fragments of kidney tissue were grafted into the right lung (Xenopus laevis) and the left lung was subsequently removed. This stimulated compensatory hyperplasia (increased mitotic rate) in alveolar tissue of the right lung and in the kidney graft. This suggests that the stimulus to compensatory hyperplasia is location-specific rather than tissue- or organ-specific. After implantation into the lung the kidney grafts are revascularized by the surrounding pulmonary supply. Damage or functional impairment of an organ usually produces a localized increase in the rate of blood flow and we therefore propose that this may lead to an increased rate of cell division in one of two ways: by permitting an increased functional capacity in the tissue concerned, or by causing a more rapid local clearance of mitotic control factors.

Notes: Myonuclei and satellite cell nuclei were differentially labelled with 3H-thymidine in uninjured skeletal muscle of young rats and then traced autoradiographically at intervals after mincing the radioactive hindlimb muscles to determine the source of regenerating presumptive myoblasts. Labelled nuclei were detected by light microscopic examination of 1-m̈ thick autoradiographs and identified by electron microscopic examination of an adjacent section. Repeated injections of 3H-thymidine during fetal and neonatal development, followed by a 4- to 5-week maturation period, resulted in labelling of 20% of the myonuclei. Satellite cells were not observed to be labelled in this series. Eight to sixteen hours after mincing, 20% of the pyknotic myonuclei were labelled, whereas none of the regenerating presumptive myoblasts appeared labelled. A single injection of 3H-thymidine administered to 18-day-old rats, followed by sacrifice within ten hours, resulted in labelling of 23% of the satellite cell nuclei. Myonuclei were not observed to be labelled in this series. Eight to sixteen hours after mincing, silver grains were detected over both pyknotic and regenerating cell nuclei. These experiments indicate that many satellite cells survive muscle injury and transplantation to become regenerating myogenic cells at a time when most, if not all, myonuclei are undergoing pyknosis.

Notes: The distribution of actin and myosin in the rat ovary at different stages of postnatal development was studied by examination of cryostat sections treated with antibodies to chicken smooth muscle (gizzard) myosin or to chicken (pectoral muscle) actin and subsequently with fluoresceinated goat anti-rabbit γ-globulins. Staining with either antiserum revealed several layers of intensely fluorescent elongated cells within the theca externa, forming a coherent band around the larger Graafian follicles. In smaller follicles, this band of fluorescent cells was incomplete, and ovaries of immature (6-day-old) rats were devoid of strongly fluorescent cells. Corpora lutea contained only scattered fluorescent cells at their circumference. Sections of mature ovaries incubated with antibodies raised against striated (pigeon pectoral) muscle myosin generated no significant fluorescence. Large oocytes stained with either anti-actin or anti-smooth muscle myosin showed a thin fluorescent band just beneath the zona pellucida, suggesting that actin and myosin are associated with the oolemma. The distribution of the two antigens in serial sections of follicles coincided, suggesting that the same cells contained both actin and myosin. It is suggested that follicular growth and maturation is attended by the development of a smooth muscle layer in the theca and that contraction of this layer in response to catecholamines and/or prostaglandins may play a role in the extrusion of the oocyte. The rǒle of contractile elements in the oocyte remains to be elucidated.

Notes: Fragments of adult rabbit lung, composed chiefly of terminal airway obtained by a trypsin digestion technique were maintained on collagen-coated cellulose sponges in Ham's F12 medium. Cell-sponge associations were examined with light microscopy, scanning and transmission electron microscopy over a period from 6 to 28 days. After an initial 24- to 48-hour period of cell migration from the airway fragment, sponge matrices became lined with cells suggestive of alveolar macrophages. After one week in culture, cysts appeared to be composed entirely of type 2 epithelial cells. These were characterized by a microvillous apical border and an elaborate junctional complex. The lumen of these cysts contained both myelin-like lamellar configurations and tubular my-elin structures such as have been described from pulmonary washings. Consistent with the age of the sponge cultures, one or more cyst types described as young, middle and late could be found simultaneously. Middle aged cysts showed signs of active secretion into the lumen. Late cysts showed changes in the epithelium comprising the cyst wall suggestive of a cell type intermediate between type 1 and type 2 epithelial cells.

Notes: The innervation of the rat Harderian gland was studied using histochemical methods for catecholamines and acetylcholinesterase (AChE). Selective denervations were performed to investigate the neural connections of this gland with various ganglia.Light microscopically the AChE-positive nerves seemed to run as thick bundles in the intertubular connective tissue. These bundles sent finer branches around the acini. The blood vessels, localized in the connective tissue septa, were surrounded by a dense plexus of AChE-containing fibres. By electron microscopy, the AChE-positive fibres were seen to terminate near the myoepithelial cells surrounding secretory cells. These fibres were also observed in contact with the blood vessels and occasionally close to the secretory cells. Fluorescent adrenergic nerves surrounded the blood vessels. Some fibres were also observed in the interlobular tissue.All the AChE-containing nerves degenerated after cutting the zygomatic nerve. On the other hand, removal of the ciliary ganglion or the superior cervical ganglion, or stereotactic coagulation of the ophthalmic nerve did not affect these nerves. The fluorescent adrenergic fibres disappeared following both removal of the superior cervical ganglion and coagulation of the ophthalmic nerve. These fibres were intact after removal of the ciliary ganglion.

Notes: Neurohistochemical techniques were used to determine the distribution of adrenergic and cholinergic nerves in the human placenta and the umbilical cord adjacent to the chorion. These morphologic studies demonstrate the absence of neural elements in these sites. The significance of these findings to the placental innervation controversy is discussed.

Notes: Human primordial germ cells (PGCs) were observed ultrastructurally in stages from their endodermal to gonadal locations. Primitive PGCs in the hind-gut epithelium of the 4-week embryo, were recognized as well demarcated cells from the neighboring cells. At the time of separation, the basal lamina of the epithelium was broken, then, through the gap so opened, the PGCs started to escape into the outer mesenchyme. In embryos at five weeks, PGCs were in the migration stage, and were found in the dorsal mesentery, at the coelomic angle and in the forming germinal ridge. In embryos at six weeks or later, almost all PGCs were accumulated in the gonad. The PGC was characterized by its large size and the large and round nucleus with conspicuous nucleolus, and by the presence of abundant glycogen particles and a considerable number of lipid droplets in the cytoplasm. Alkaline phosphatase activity was demonstrated selectively on the plasma membrane of the PGC. The shape of PGC was irregular, often had pseudopodia in PGCs in the separation and migration stages, suggesting their amoeboid movement in vivo, but was generally round or elliptic in PGCs in the settlement stage. The PGC was usually surrounded by and in close association with adjacent somatic cells.

Notes: Ovaries from guinea pigs of different ages have been examined by light microscopy. Serous cysts measuring between 1 mm and 2.5 cm are present in all animals over one year of age. The cysts are fewer in number and size at six months, are least developed at ten weeks and are not apparent in the nearterm fetus. The cysts arise from the rete ovarii. They are lined with a simple cuboidal to columnar epithelium composed of cells bearing solitary cilia or tufts of cilia. The structure and origin of these cysts is discussed and compared with what is known of the human serous cystadenoma.

Notes: Rana pipiens tadpoles were injected with 3H-thymidine at different stages to label basal cells of the horny beaks, the keratinized oral specializations of many anuran tadpoles. Cells in the column and sheath of the beak are derived from the basal layer. Movement of the labeled cells in beak columns can be traced, permitting calculation of the rate of cell addition into the columns. With the counts of the total number of column cells and cell addition rate, the rate of apical cell loss from a column throughout the larval stages can also be calculated. The rate of cell addition decreases steadily from stage III through stage XX, in all portions of both beaks. The rate of change of these column cell addition rates is similar among middle and lateral portions of both upper and lower beaks. There is a relatively constant rate of cell loss until stage XII or XIII. It then increases sharply, and the beaks are completely lost at stages XIX or XX. The longer, lower beak columns have a much faster cell loss rate than do the upper beak columns at late larval stages. Life spans for cells that move to the sheath may be about one-half those for cells that enter the beak columns. It is suggested that thyroid hormone accelerates the rate of column cell loss.

Notes: Normal or pre-denervated extensor digitorum longus (EDL) muscles were successfully grafted in place of the contralateral EDL muscles in cats. Histological preparations of 57 grafts were examined at intervals from 4 to 170 days after transplantation. The morphological sequence of events in a cat muscle graft is similar to that in the rat except that the time course of regeneration is considerably slower. Surviving original muscle fibers form a thin rim at the periphery of the graft. Beneath this rim, the original muscle fibers undergo fragmentation, starting near the periphery and progressing toward the center. Regenerating muscle fibers take the place of the degenerated muscle fibers. In pre-denervated grafts, the last of the original muscle fibers in the center of the graft have broken down by 41 days whereas in normal muscle grafts the original muscle fibers in the central region persist until 50 days. The main difference between grafts of normal and pre-denervated muscles is the rate of breakdown of the original muscle fibers. Long term grafts of both groups are morphologically very similar.

Notes: During a study on the fine structure of different regions of the reproductive tract of the ground squirrel, Citellus lateralis, cells possessing cytological features associated with steroid production were observed among the ductuli efferentes. Comparison of the morphology of these cells with that of Leydig and adrenocortical cells, demonstrated that they resembled Leydig cells in many anatomical details. Further evidence for the similarity of the cells associated with the ductuli efferentes to Leydig cells emerged during sexual regression, when both types of cells underwent the same regressive changes in organization and morphology. This suggests that the cells present among the ductuli efferentes are active in the synthesis of androgenic hormones, and possibly play a role in the endocrine control of reproduction in this species of ground squirrel.

Notes: Polymorphonuclear leukocytes (PMN's) incubated three to eight minutes at 37°C in medium containing 1 × 10-6M of the ionophore antibiotic A23187 released their cytoplasmic granules into the extracellular medium. Transmission electron microscopy of treated cells showed microfilament bundles extending between adjacent granules within the cytoplasm and between granules and the plasma membrane. Tiny dense projections (beads) 8-12 nm in diameter were observed along segments of the cytoplasmic surface of the plasma membrane with a periodicity of 20-30 nm. These beads were observed on the plasma membrane only in the vicinity of intra- or extracytoplasmic granules. The structural relationships of the beads with the plasma membrane micro-filaments suggest they play a role in the process of ionophore-induced granule release from polymorphonuclear leukocytes.

Notes: A method is presented by which fresh anatomical material may be preserved in a dry state while maintaining joint function and motion. This method could be likened to the tanning of the tendons and ligaments directly on the bones. Mastery of this technique has allowed the preservation of not only the tendinoskeletal system, but also the nervous, vascular, and endotracheal systems as well. While some skill in dissection is necessary, a large amount of the dissection can be done by chemical methods. The actual processing of the specimen is easily done with materials that are readily available. When finished, the specimen is dry, flexible, essentially odorless, and extremely durable. This method can be utilized for material ranging from simple osseous specimens to highly sophisticated dissections. These specimens have been found to be of exceptional advantage in teaching by demonstrating function as well as spatial relationships.

Notes: The differentiation of the mitral cell perikaryon in postnatal rat olfactory bulb was studied with the light and electron microscope. At birth the mitral cell was distinguishable and occupied a definitive position in the mitral cell layer. The cell contained a large oval nucleus surrounded by a thin rim of cytoplasm. Ribosomes, free and clustered, were scattered in the cell cytoplasm. Rough endoplasmic reticulum was relatively scarce. The Golgi complexes were made up of stacks of smooth-surfaced cisternae and associated vesicles. In certain cases the Golgi complexes projected into cellular processes. Mitochondria were present in all regions of the cytoplasm and contained well developed cristae. At the end of the first week, the mitral cell had developed significantly in size, and the cytoplasm contained well-developed rough endoplasmic reticulum. The Golgi complexes were made up of several stacks of smooth-surfaced cisternae with the association of vesicles and electron dense bodies. The apical dendrites o mitral cells at this periiod had increased significantly in length. Subsequently, during the second and third week, the rough endoplasmic reticulum and Golgi complexes became well developed. Associated with the Golgi complexes were electron dense lysosomal bodies. At three weeks and in older cells it was observed that dense lysosomal bodies. At three weeks and in older cells it was observed that dense lipofuschin granules increased significantly. It is suggested that the mitral cell matures and differentiates earlier than cells in the cerebral cortex.

Notes: A method was developed to alter surgically the bursa of the bitch to allow sequential observation of the ovary by means of laparoscopy. Adult anesthetized female dogs of three breeds underwent laparoscopic examinations in which each natural bursa slit was lengthened 1.0-1.5 cm using laparoscopic hook scissors in conjunction with electrocautery. The ovary could be effectively and repeatedly exposed for laparoscopic viewing through the lengthened bursa slit. Neither Laparoscopic surgery nor frequent routine laparoscopic examinations of the reproductive organs had an effect on subsequent ovarian activity. All dogs subjected to the laparoscopic ovarian exposure technique exhibited estrus behavior, follicle development, ovulation and corpus luteum formation.Preovulatory follicular development was detected as early as 11 days before ovulation with the most rapid follicular maturation occurring two to three days before ovulation. Follicular collapse and extensive hemorrhage at the rupture site during ovulation did not occur. Release of the cumulus mass from a vesicular follicle was observed in one bitch. The cumulative mass adhered to the follicular wall for an indefinite time following ovulation.The structural size of the bitch corpus luteum was maximal four to eight days following ovulation. Corpora lutea remained visible on the ovarian surface for 16 weeks. No differences in cyclic ovarian morphology were observed among the three breeds of dogs studied.

Notes: Ventral pharyngeal outpocketings and ultimobranchial out-pocketings from the 14-day-old fetus of the Fischer rat were isolated before they fused to form thyroid glands. The outpocketings were implanted into different kidney capsules of adult male Fischer rats, and were allowed to grow for several months. Transplants were then excised and examined by electron microscopy. The ultimobranchial outpocketing gave rise to two types of follicles. One contained ciliated cells, cells with an abundant agranular reticulum, U cells with basal hemidesmosomes, and two types of cells with secretory vesicles. The other contained C cells separated from the follicular lumen by a single flat fiber-con-taining cell. The ventral outpocketing formed typical thyroid epithelium making up the usual thyroid follicles differing from follicles in the thyroid in situ by the absence of C cells. These follicles were functional as determined by autoradio-graphic studies with 125I but differed from thyroid follicles in situ with respect to size distribution. The results suggest that (1) in the adult thyroid gland the C cell in the usual follicle is an ultimobranchial contribution and (2) the so-called ultimobranchial follicle is ultimobranchial in origin but that the typical thyroid epithelium in mixed follicles of U cells and typical thyroid epithelium is a ventral contribution. The reason for the absence in the thyroid gland in situ of the variety of other cell types observed in ultimobranchial transplants is unknown.

Notes: In an attempt to localize the site of remodelling of collagen in the periodontal ligament of the continuously erupting mouse incisor a radioautographic and stereologic investigation was undertaken. Grain distributions in radioautographs for light microscopy were studied at various time intervals after administration of [3H]-proline. The distribution of rough endoplasmic reticulum cisternae in fibroblasts and the incidence of collagen phagocytosis across the ligament were studied by means of stereologic methods at the electron microscopic level.No significant differences were found in half lives of [3H]-labelled substances among the various regions across the ligament. Roughendoplasmic reticulum cisternae within fibroblasts were distributed more or less uniformly throughout the entire width of the ligament. Analysis of the distribution of collagen phago-cytosis, however, revealed that in the midregion of the ligament the amount of phagocytosed collagen was approximately four times as high as in the area adjcent to the tooth and about nine times as high as in the alveolar compartment.It is concluded that synthesis and turnover of total protein occurs throughout the periodontal ligament but that remodelling of collagen predominantly takes place in an intermediate area of the ligament.

Notes: A study of the development of hemopoiesis in the Mongolian gerbil (Meriones unguiculatus) was conducted in order to determine the temporal sequence, the organs involved and the cytology of blood cell formation in this species. Hemopoiesis in the intrauterine life of the gerbil can be divided into four phases based on the site of blood cell formation: (1) the vitelline phase, (2) the hepatic phase, including thymic histogenesis, (3) the splenic phase and (4) the medullary phase, with the development of secondary lymphoid tissues. At the onset of each of these phases a blast-like cell was identifiable in each hemopoietic organ which, because of its morphology and its presumed multipo-tentiality was classified as a “lymphoid cell.” In the yolk sac phase (gestational day 12) two generations of erythrocytes, a primitive and a definitive, are formed. The liver is by day 15 erythropoietic and megakaryopoietic, but later, a few gran-ulocytes are also found in its extravascular compartment. The thymus is exclusively lymphopoietic from the appearance of its earliest cells on day 15. Splenic hemopoiesis is initiated with the presence of lymphoid cells (day 20) followed later by the appearance of morphologically identifiable blood cell lines. Early normoblastic and granulocytic activity begins in the marrow cavities on day 23, though the marrow is not considered to be a source of circulating blood cells during fetal life. Lymph node histogenesis occurs during the last four days of gestation, first in the cervical region and then in other parts of the body. The finding of undifferentiated lymphoid cells in all organs at the initiation of hemopoiesis and in the peripheral blood throughout gestation is discussed in light of the migratory theory of hemopoiesis.

Notes: The basement membranes in the exocrine pancreas were examined by routine electron microscopy, by fixation for electron microscopy with a glutaraldehyde-tannic acid solution and by staining for ultrastructural demonstration of mucosubstances with the dialyzed iron, high iron diamine, ruthenium red and Concanavalin A-horseradish peroxidase procedures. The basement membranes are considered from morphologic and histochemical observations to consist of an inner lamina lucida, an intermediate lamina densa and an outer lamina diffusa. Sulfated mucosubstance was found in the lamina diffusa of the basement membrane of all epithelial cells and capillary endothelial cells but was encountered in the lamina lucida of the duct cells exclusively. Bridging structures, presumably polypeptides, were also seen connecting the lamina densa and the basal plasma membrane in specimens fixed with the glutaraldehyde-tannic acid solution. The findings demonstrated that the histochemical and morphological qualities of the basement membrane are uniform for a given cell type but differ considerably among the several cell types in the pancreas.

Notes: Cytological examination of the rat incisor enamel organ with the light and electron microscope revealed a small number of ameloblasts which contained two and sometimes three or more nuclei per cell. A multinucleate ameloblast usually contained two vertically apposed nuclei situated near the base of the cell. A narrow cytoplasmic band was interposed between adjacent nuclear envelopes. The apical nucleus was often the more elongated of the two nuclei and it fitted a convexity or a concavity within the more basally positioned nucleus. In serial sections examined with the electron microscope no connections were observed between the nuclei. In animals injected with 3H-thymidine instances of multinucleate ameloblasts were found within the advancing front of labeling where only one of the nuclei contained label. Finally, quantitative analysis by nuclear counting established that multinucleate ameloblasts were 60 times more frequent within the maturation zone as in the secretory zone of amelogenesis. As well, the numbers of multinucleate ameloblasts increased progressively in the course of the maturation stage. It was concluded that multinucleate ameloblasts increase with cell age and likely arise by the process of cell fusion.

Notes: Single, isolated pancreatic islets of mice and rats were incubated for varying time intervals (0.5-60) minutes with high (300 mg%) and low (50 mg%) levels of glucose. The structural integrity of islets decreased progressively with time regardless of glucose concentration. Degeneration of islets was greatest after 60 minutes of incubation. The total amount of insulin released from cytologically intact mouse islets incubated with high glucose levels was always greater than that with low glucose except following 30 seconds of incubation when no difference was observed. Peaks of insulin secretion noted after 2 and 15 minutes of incubation were correlated with light microscopic and fine structural changes indicative of active secretion in β-cells, i.e., degranulation, granule margination. At 5 and 30 minutes of incubation many β-cells contained enlarged Golgi zones and abundant profiles of swollen rough endoplasmic reticulum containing pale, amorphous granular material presumably indicating insulin synthesis. Emphasis is placed on the desirability of correlating physiological and biochemical studies of isolated pancreatic islets with cytologic examination.

Notes: Recently, radioautographic studies have shown that cell coat glycoproteins are transported to the cell surface by vesicles both in the amoeba (Flickinger, '75) and in the epithelial cells of the ascending colon of the mouse (Michaels and Leblond, 1976). In the current morphological and cytochemical study of the surface epithelial cells of the rat ascending colon, it is shown that filamentous material, resembling the cell coat, is contained in saccules toward the mature face of the Golgi apparatus and in vesicles close to the apparatus and near the terminal web. The vesicles are limited by a unit membrane composed of asymmetric osmiophilic leaflets and similar to the plasma membrane. When stained by the periodic acid-chromic acid-silver methenamine technique, silver was precipitated on the cell components containing the filamentous material indicating the presence of glycoproteins. Narrow invaginations from the cell surface that may correspond to vesicles undergoing exocytosis were also positive for glycoproteins. The distribution of the filamentous material that was glycoprotein positive parallels the pathway followed by material that had been found to be labeled with a tritiated glycoprotein precursor (3H-fucose) in the epithelial cells of the ascending colon of the mouse. It is suggested that the system of vesicles in the rat colon cells is acting in a manner similar to the vesicles in the mouse cells to transport cell coat glycoproteins from the Golgi apparatus to the cell surface.

Notes: A spaced serial section, electron microscopic study of the extensor digitorum longus of the 2-day-old rat was undertaken to determine whether all cells wedged between the basement membrane and the sarcolemma of a muscle fiber, regardless of their morphology, were true myosatellite cells. The muscle was relatively immature, containing groups of cells enclosed in a common basement membrane (clusters) and exhibiting primitive myoneural junctions. Cells of the muscle line included in the clusters were primary fibers, satellite fibers, myotubes in different stages of development and myosatellite cells. In single ultrathin sections, some early myotubes lacked myofilaments and were difficult to distinguish from myosatellite cells. Spaced serial sections revealed that all myosatellite cells have heterochromatic nuclei and a high nuclear/cytoplasmic ratio, and that all pale staining nuclei were found in cells with a lower nuclear/ cytoplasmic ratio, containing variable quantities of myofilaments. In addition to the cells of the muscle line, mast cells, fibroblast-like cells and other “unclassified” cells were entirely or partially enclosed within the cluster's basement membrane. This study demonstrated that location alone or location and the morphological appearance of cells in a single ultrathin section failed to provide sufficient criteria to properly classify all of the cells found in neonatal muscle clusters.

Notes: The present work reports a tridimensional reconstruction of the intratesticular seminal pathway in the hamster as studied with light microscopy of serial sections of the proximal pole of the testis. It has three portions: a “terminal segment” of the seminiferous tubules; the tubuli recti with three parts; and the rete testis with two parts, intratesticular and extratesticular.Experiments using ductuli efferentes ligation show a marked dilation of the rete testis and tubuli recti without a corresponding dilation of the seminiferous tubules. This may be due to a valve-like action of the cells in the terminal segment of the tubules.

Notes: The interstitial tissue of the testis of the nine-banded armadillo is composed of blood vessels, clusters of Leydig cells, the usual connective tissue elements, and a network of lymphatic sinusoids. The endothelial walls of the sinusoids are separated from the peritubular contractile cell layer surrounding the seminiferous tubules by a thin layer of collagen. The peritubular contractile cell is characterized by filaments and dense bodies within the cytoplasm, whereas the endothelial cells lack these structures. Within each cluster, several Leydig cells surround one or more blood vessels. Adjacent Leydig cells are joined by 2- to 3-nm wide gap junctions and desmosome-like specializations. The Leydig cell is polygonal in shape with an ovoid nucleus. The cell is characterized by an abundance of smooth endoplasmic reticulum which appears as sheets of membranes, concentric whorls around vacuoles, and a random tubular network. Only a few short cisternae of rough endoplasmic reticulum are observed. Centrioles are closely associated with the Golgi apparatus. Rod-like mitochondria with tubular cristae are scattered throughout the cytoplasm. In addition, the cells contain vacuoles resulting from lipid extraction, filaments, microtubules, and glycogen. The surfaces of the cell exposed to the intercellular spaces exhibit numerous pinocytotic vesicles and cell processes which indicate active movement of material across the plasma membrane. In comparison to other mammalian species, the ultrastructural organization of the interstitium and the fine structure of the Leydig cell of the armadillo resemble those of the guinea pig.

Notes: The combination of a progestin and androgen has received attention as a possible male contraceptive. The progestin is thought to reduce gonadotropin release and suppress spermatogenesis, while the sex accessory organs and male characteristics are maintained by the simultaneous administration of testosterone. In the present study, the histology and ultrastructure of parts of the male reproductive tract of rats treated with medroxyprogesterone (Provera, Upjohn) (1 mg/100 g body weight/day) alone and combined with testosterone (15, 30, or 100 μg/100 g/day) were studied following treatment for up to 16 weeks. The testes and epididymides of rats administered Provera alone or Provera and testosterone weighed less than those of control rats. The weights of the accessory glands of rats treated with Provera were greatly reduced; it was possible to maintain them at approximately control levels by simultaneously administering sufficient testosterone (100 μg/100 g body weight/day). The fertility of some of the animals was tested by caging them with female rats, and none of the treated rats tested in this way was fertile.Similar microscopic alterations were present in the testes of animals administered Provera alone or Provera and different levels of testosterone. Spermato-gonia, spermatocytes, and early spermatids were abundant in treated rats and did not show ultrastructural changes. However, many degenerating or necrotic spermatids of the cap phase (approximately stages 6-7 ) and later were present. Late spermatids of the acrosome and maturation phases were rare. Some necrotic spermatids were surrounded by Sertoli cells, and parts of spermatids lay within lysosome-like structures in the cytoplasm of Sertoli cells. Many large lipid droplets were also present in Sertoli cells of treated rats. Leydig cells were smaller in treated animals than in control rats. The results suggest that germ cells can develop up to cap phase spermatids but then undergo degeneration. These alterations in spermatogenesis may be responsible in large part for the antifertility effect of the progestin and androgen combination.Some rats were permitted to recover following the end of treatment. The microscopic appearance of the testis returned to normal within three to six weeks, although epididymal alterations persisted in some animals six weeks after the end of treatment. By 9 to 12 weeks after the end of treatment the reproductive organs had a normal microscopic appearance in all the rats studied.

Notes: A dependable method for freeze-drying tissues for electron microscopy has been developed. Thin slices of fresh tissue were frozen by bringing them into direct contact with a polished copper bar at liquid nitrogen temperature. The tissue was transferred to a copper specimen block equipped with a thermocouple and heating circuit for accurate control of the environmental temperature of the tissue, and evacuated in a glass freeze-drier using clean high vacuum techniques for keeping the system free of hydrocarbons. The tissue was dried by increasing the temperature of the specimen block 10°C each hour while monitoring the rate of water removal from the tissue with a partial pressure analyzer. The dry tissue was fixed with OsO4 vapor, vacuum embedded in a low viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Tissue processed in this manner exhibits excellent morphological preservation at both cellular and organellar levels without prefixation or the use of cryoprotective agents. The results of the experiments using the partial pressure analyzer indicate that small blocks of tissue can be dried in a short time at low temperature.

Notes: Young adult male rats were administered medroxyprogesterone (Provera, Upjohn) alone and in combination with testosterone, as has been done to inhibit male fertility. The histology and fine structure of several segments of the epididymis, the ventral prostate, and the seminal vesicle were studied at intervals after treatment for up to 16 weeks. The epididymides of treated animals weighed less than those of control rats. Microscopic alterations in the epididymis were similar in rats treated with Provera alone and in those animals that received Provera and testosterone, but the changes varied with the segment of the epididymis. In the middle segment in the caput epididymidis, the normally abundant luminal sperm were absent but the epithelium retained its normal ultrastructural features. In the terminal segment in the cauda epididymidis, different changes were observed in the proximal and distal portions. In the proximal cauda epididymidis, the lumen was small, irregular in outline, and virtually devoid of sperm. The light cells of the epididymal epithelium in the proximal cauda contained extremely large numbers of dense bodies resembling lysosomes, which occupied most of the supranuclear and basal cytoplasm. In contrast, in the distal part of the cauda epididymidis, the epithelium had a normal appearance but the lumen was filled with debris, sperm, and spherical masses of cytoplasm that were apparently derived from germ cells. It is suggested that the clearing of the lumen of the proximal cauda epididymidis may reflect the greater activity of light cells of the epididymal epithelium in that region. Although alterations in spermatogenesis may be most important in the antifertility effect of progestin and androgen, these alterations in epididymal sperm and epithelium may also play a role.The weights of the prostate and seminal vesicles of rats treated with Provera (1 mg/100 g/day) were greatly reduced compared to those of control rats. Although there was considerable variation, in many specimens treated with Provera alone the epithelium of the prostate showed a change from a columnar to a cuboidal or squamous shape, and there was a reduction in the size and abundance of organelles involved in the formation of secretions. The microscopic structure of the seminal vesicle of rats treated with Provera was less severely affected than the prostate. Although the seminal vesicle epithelium of Provera-treated rats was generally not as tall as in control animals, the cells possessed parallel cisternae of rough endoplasmic reticulum, secretory vacuoles, and an active-appearing Golgi apparatus, suggesting that they continued to be able to form secretions in the presence of Provera. The weights of the sex accessory glands were maintained at control levels by the administration of testosterone, 100 μg/100 g/day, along with the Provera. A normal fine structure was present in the epithelium of both the prostate and seminal vesicle of rats administered this amount of testosterone in addition to Provera. Lower doses of testosterone (15 or 30 μg/100 g/day) were insufficient to maintain normal weight or ultrastructure of the sex accessory glands in the presence of Provera.

Notes: Morphological changes in cutaneous nerve endings were investigated electron microscopically in patients suffering from certain kinds of urticaria with associated itching and in normal skin in which wheals and local itching were induced either by application of nettle hairs or by intracutaneous injections of a timothy pollen extract. Skin samples were obtained with a high speed dermal punch without anesthesia from the wheal areas.It was found that some subepidermal free nerve endings derived from nonmyelinated nerve fibers (penicillate endings) showed accumulations of extracytoplasmic glycogen which was localized in the distended spaces between the axolemma, the Schwann cell membrane and the nerve basement membrane. In some cases, the glycogen was found to be so abundant that it occupied most of the cross sectional area of the ending. No morphological changes were observed in the pappilary endings, in nerve endings of the hairs or in the autonomic terminals. The conducting segments of all cutaneous nerve fibers showed normal morphology.The unusual morphological changes that occur in some penicillate nerve endings during the wheal development indicate that these endings are involved in the skin reaction and therefore they may be the specific end organs that are associated with itch, at least in urticaria.

Notes: Concanavalin A (Con A), a lectin binding to mannosyl and glucosyl residues of glycoproteins and glycolipids, was used to study the appearance of carbohydrate-rich cell surface material on the olfactory placode and nasal processes which contribute to formation of the primary palate. In vivo incorporation of 3H-thymidine was also used in an attempt to correlate changes in labeling index with formation of the olfactory placode and nasal processes.The cell surface of the early frontonasal epithelium binds Con A very little, if at all, but Con A binding was observed when the olfactory placode could be identified as a plate of cuboidal cells that exhibited a reduced labeling index. During the period of formation of the nasal processes, Con A binding was observed on the facial epithelium including the presumptive contact region. There was also a decline in the labeling index throughout primary palate formation.This study provides three criteria by which the olfactory placode can be identified: a morphological change of placode cells to a cuboidal shape, a synthesis or rearrangement of surface coat material that binds Con A, and a reduced labeling index.

Notes: Reticular and collagenous fibers stain differently when subjected to ammoniacal silver reduction. A variety of tissues were subjected to such a “reticulin” technique and the association of reaction product with intercellular connective tissue elements was studied with the electron microscope. The reaction with reticular fibers was primarily associated with the interfibrillar matrix, and was globular in form having a wide variety of particle sizes. Conversely, in dermal collagen the unit fibrils were stained rather than the interfibrillar matrix. The precipitate was punctate in form and was associated with the cross striations of unit collagen fibrils. Large microfibrils also reacted positively with the stain, imparting a faint periodicity. Basement membranes were stained uniquely. The underlying plasmalemma and the lamina densa were heavily stained with silver while the lamina lucida was relatively unstained. The unit fibrils of the lamina reticularis stained in the same manner as dermal unit collagen while the ground substance remained unstained. This represents a clear distinction between the argentophilic characteristics of collagenous fibers, reticular fibers, and basement membranes.

Notes: Mice fed a high cholesterol-cholic acid diet for two to six months develop gallstones; these were studied by transmission electron microscopy after glutaraldehyde-digitonin fixation. Examination of the contents of mouse gallbladders presents views of layered structures and surrounding amorphous material. We interpret these images of gallstones to suggest that they may arise by cohesion of material rich in cholesterol to form more ordered structures. Gallbladder contents of mice fed the diet for five to six months were found to contain occasional crystals and rectangular areas similar to those observed in thin sections of human gallstones (unpublished observations). Recent findings that human gallstones can be dissolved with chenodeoxycholic acid are discussed, with reference to their applicability to studies of gallstones in mice.

Notes: An electron dense material is found in the extracellular space of the nervous system of Aplysia, a marine mollusc whose ganglia are widely studied by neurobiologists. This material appears to consist of irregular electron-dense granules, with diameters of approximately 600 Å. This material is found between the glial cells that surround the neuronal perikarya. It is not found in other regions of the nervous system. Because it is found in the ganglion cell layer of the nervous system and because the neurons contain what may be the precursor of this substance, the electron-dense material is regarded as most probably being a neuronal product. The importance of this material is that it is one of the few examples of visible structure in the extracellular space of the nervous system.

Notes: Scanning electron microscopy of permanent tooth buds of the monkey confirmed that mineralizing interrod enamel surrounds Tomes' processes on three sides, forming pits that restrict enamel rod formation. The forming face of the enamel rod, which is the floor of the pit, angled toward the tooth surface at the apical edge of the pit, the side nearest the cervical region of the tooth. Consequently, the apical edge of each pit was the only site where both rod and interrod enamel were formed at the nascent tooth surface.The ameloblasts had two secretory surfaces. One was the microvillous surface of the short Tomes' process abutting the forming face of the enamel rod. The other surface, closer to the ameloblast, was between Tomes' processes, abutting the crests of interrod enamel which formed the pits. At each site forming enamel crystallites had specific orientations.Due to the angle of the forming face of the rod and the short Tomes' process, crystallites with both rod and interrod orientation form at the same time and the same plane within the apical (cervical) margin of each rod. It is hypothesized that indistinct boundaries between rod and interrod enamel at the apical margin of each rod are due to both secretory surfaces of ameloblasts secreting at the same time and at the same site.