Ultrastructure of the interstitial cells of Leydig, stimulated and unstimulated

The interstitial cells of Leydig which lie in small groups or individually between the seminiferous tubules of the testes, or at the hilum in the ovary, are known to be active in production of androgenic substances, as well as being sensitive to the influence of various trophic hormones. Among the hormones known to be produced by these cells are testosterone, dihydrotestosterone, and estradiol. Responsiveness of the function of the Leydig cells has been demonstrated with luteinizing hormone (LH), growth hormone, follicle stimulating hormone (FSH), and probably estrogen as well as prolactin. Human chorionic gonadotrophin also may have a marked effect. Attempts to correlate the cytologic appearance of Leydig cells with various states of stimulation have revealed a number of ultrastructural appearances. Since a spectrum of cellular morphology is apparent, both in the normal and in altered physiologic states, it is hazardous to ascribe a particular ultrastructural variation to the influence of a given hormonal stimulus. Nevertheless, in normal males, three types of cells can frequently be seen: fusiform cells with ovoid nuclei, small aggregates of smooth endoplasmic reticulum (SER), and variable amounts of cytoplasmic filaments, probably representing resting cells, since they are most abundant in pre-pubertal males; light cells, the most frequent type, with well developed SER, scant rough endoplasmic reticulum (RER), and mitochondria which vary in size and shape, contain abundant lipid and frequent lipochrome deposits; and dark interstitial cells which are variable in number, derive their density from stacks of tubular SER, and may represent merely an altered response to fixation or an involutional form. In addition to immature cells and normal mature cells, two additional cell types are described in various primary testicular disorders: abnormally differentiated Leydig cells with features such as grouped mitochondria, whorls of endoplasmic reticulum, absent or fragmented Reinke's crystals and paracrystalline arrays and deficiency of lipid droplets as well as masses of microfilaments; and a multivacuolated cell type characterized by swelling of cytoplasmic organelles and absence of Reinke's crystals or paracrystalline arrays. It is also clear that the interstitial cells respond in systemic diseases, are injured by alcohol ingestion, and show involutional changes in aging. While these ultrastructural changes are now well documented, there is still a need to correlate them exactly with the various stimuli which may affect testicular function.     

Leydig cell tumor of the testis: a case diagnosed by fine-needle sampling without aspiration with histologic, immunohistologic, and electron microscopic analysis.

Fine-needle sampling without aspiration was performed in a patient with a testicular mass. The cytologic diagnosis was consistent with Leydig cell tumor. Cytologic features included abundant grey-blue cytoplasms with spherical or oval nuclei in May-Grünwald-Giemsa-stained smears. Intranuclear inclusions were observed but no Reinke's crystals were detected. Histologic findings confirmed the diagnosis and tumor cells were positive for vimentin. Electron microscopic analysis of the tumor showed abundant smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae but no Reinke's crystals.     

Ultrastructure of immature leydig cells in the human prepubertal testis

The cellularity of the human prepubertal testicular interstitium has not been well studied at the ultrastructural level. In this study, testicular biopsies were obtained from 35 boys aged three to nine years and examined by electron microscopy to clarify and quantitate the cell types present during the prepubertal period. The prepubertal testicular interstitium is found to consist of immature Leydig cells (9%), primitive fibroblastic cells (63%) (intertubular in location), and attenuated peritubular fibroblasts (28%). The primitive fibroblastic cells and peritubular fibroblasts appear closely related, being distinguished mainly by shape and location. The immature Leydig cell type contrasts with the fibroblastic cell types by exhibiting an irregular nucleus with relatively little heterochromatin. The most impressive cytoplasmic feature is the moderate to extensive development of smooth endoplasmic reticulum in the form of anastamosing tubules. In contrast, the rough endoplasmic reticulum is not well developed. Other cytoplasmic characteristics are the highly developed Golgi elements and occasional lipid droplets and lysosomes. Glycogen is also often present and is generally found in those cells that do not contain a welldeveloped smooth endoplasmic reticulum. The ultrastructure of the immature Leydig cell is compared with that of the mature fetal and adult Leydig cells. Although generally found in small clusters between tubules, these cells are often attenuated and closely associated with the seminiferous tubules. Occasional intermediate cell morphologies suggest a relationship between the primitive fibroblasts and immature Leydig cells. The presence of small cells exhibiting a steroid-producing morphology, classified as immature Leydig cells, in the prepubertal testicular interstitium is an interesting finding and is in accordance with earlier studies on nonhuman mammals. It is unknown whether these cells are remnants of the fetal Leydig cell population or have differentiated neonatally from the primitive fibroblastic cells. It is suggested that the immature Leydig cells are the progenitors of the adult Leydig cell population.     

Testicular feminization of the mouse: paucity of peroxisomes in Leydig cell of the testis.

The testes of mice with the X-linked testicular feminization (Tfm/y hermaphrodite) mutation are very small and cryptorchid. The spermatogenesis in the adult Tfm/y hermaphrodite mouse testes is arrested, and the testosterone biosynthesis is significantly reduced due to deficiency of 17-keto-steroid reductase, the enzyme essential for the conversion of androstanedione to testosterone. In this study the distribution of peroxisomes in the Leydig cells of adult Tfm/y hermaphrodite mice was investigated because of the suggestion that peroxisomes may participate in lipid metabolism and/or androgen biosynthesis is steroidogenic cells. Aldehyde-fixed testicular tissue of Tfm/y hermaphrodite mice was processed for the cytochemical localization of peroxisome catalase to facilitate identification of these organelles in the Leydig cells. Testes from Blo/y and CSa strain normal adult mice served as controls. Testicular Leydig cells of normal adult Blo/y and CSa mice contained abundant smooth endoplasmic reticulum (SER) in the form of complex interconnected tubules and double-walled membranous vesicles. Numerous peroxisomes, often in continuity with SER channels or in close association with lipid droplets, were observed in Leydig cells of normal males. In contrast, the peroxisomes in the Leydig cells of adult Tfm/y hermaphrodite mouse testes were either undiscernible or greatly reduced in number and size. SER in these cells was sparse, whereas mitochondria were numerous. In addition, abundant clusters of lipid droplets were encountered in a majority of Leydig cells of Tfm/y hermaphrodite mouse testes. Peroxisome and SER paucity in Leydig cells of Tfm/y hermaphrodite mice may be a reflection of reduced testosterone production. Whether excessive accumulation of lipid in the Leydig cells of Tfm/y hermaphrodite mouse testes is due to reduced utilization of cholesterol for the biosynthesis of testosterone or to impaired lipid metabolism due to reduction in peroxisome population in these cells remains to be ascertained.     

An extra-pancreatic direct effect of streptozotocin on the hypothalamo-hypophyseal-testicular axis in the rat

Streptozotocin induced diabetes mellitus in 4 out of 6 male rats. On the other hand, all 6 drug-injected animals had decreased serum testosterone values. The Leydig cells from 6 control rats and all streptozotocin-injected animals were characterized by moderate numbers of mitochondria, few lipid droplets, and an abundance of smooth surfaced endoplasmic reticulum. Quantitative analysis of numbers of mitochondria and lipid droplets revealed no differences between control and streptozotocin-injected animals. However, there were smaller mitochondria, larger lipid droplets and a trend toward fewer cells with dilated profiles of smooth endoplasmic reticulum in all drug injected animals. These cytological and biochemical findings suggest that diabetes per se had little direct effect on Leydig cell function. However, taken together with previous studies, these observations suggest that streptozotocin may directly affect the hypothalamo-pituitary-testicular axis possibly at the level of LHRH-secreting neurons of the hypothalamus.     

Ultrastructural evidence of mature Leydig cells and Leydig cell regression in the neonatal human testis

The neonatal period in male development is characterized by an acute rise in serum testosterone, which peaks at 2 to 3 months of age. The purpose of this study is to examine the neonatal human testicular interstitium at 4 months for evidence of Leydig cell maturation, as well as any morphological criteria relating to the fate of Leydig cells during this period, specifically, for signs of cell regression. Leydig cells are described with impressive development of the steroid secreting apparatus, which are consistent with the mature Leydig cells found during early fetal development and in the adult. The outstanding feature of these cells is the "organelle association" of extensive, anastamosing tubules of smooth endoplasmic reticulum (SER), pleomorphic mitochondria with a component of tubular cristae, and abundant microperoxisomes associated with the SER. Well-developed Golgi elements, regionalized RER, and diverse cell inclusions are also characteristics of these cells. Reinke crystals and paracrystalline inclusions are absent. Gap junctions are common in this system and are notable in the asymmetric nature of the adjacent cytoplasmic components. These findings provide a morphologic correlate to the reported neonatal phase of testosterone production in man. Intermediate forms of Leydig cells are described with "organelle associations" including decreased SER with increased lipid droplets, and decreased SER with prominent cytoplasmic filaments and/or dramatic mitochondrial changes supportive of mitochondrial involution. Cells consistent with immature Leydig cells are also present. The rather impressive diversity in cell morphology present during this time frame of 4 months, slightly past the peak in testosterone production, provides evidence of Leydig cell regression and a continuity of the mature neonatal Leydig cells with the immature Leydig cells of childhood (Prince, 1984). There is also some evidence of cell degeneration. Although the developmental history of Leydig cells has been described for years as biphasic, it is time to view Leydig cell development in man as a triphasic event, fetal, neonatal, and pubertal.     

A fine-structural study of interstitial cell changes in the testes of Necturus maculosus during a portion of the annual cycle,and possible evidence for local feedback control by seminiferous epithelium

The developmental stages of the interstitial cells of Leydig in Necturus maculosus were studied in testes of animals obtained in November and December. As a consequence of the caudal to cranial progression of the wave of spermatogenesis, samples obtained from cranial, central, and caudal portions of these testes exhibit regional differences in the morphology of their Leydig cells. In these samples, fibroblast-like stromal cells with large, dense, elongated nuclei and thin sheets of cytoplasm surround the cranial seminiferous lobules that usually contain spermatozoa. Such stromal cells possess a small juxtanuclear Golgi apparatus, profiles of smooth and rough endoplasmic reticulum, and mitochondria with tubular and shelf-like cristae. In the central area of the testis, interstitial cells with oval nuclear profiles and a finer chromatin pattern surround degenerating seminiferous lobules. The abundant cytoplasm of these cells contains predominantly smooth endoplasmic reticulum interspersed with a few lipid droplets. These cells contain mitochondria that are packed with tubular cristae. In the caudal part of the testis, the Leydig cells have a round nucleus with finely dispersed chromatin. Numerous lipid droplets accumulate in the ample cytoplasm, which is filled with smooth endoplasmic reticulum. The juxtanuclear Golgi apparatus becomes enlarged; associated with it are vesicles with an electron-dense content. The cristae of the mitochondria are more numerous and have tubules of 50-nm diameter mixed with arrays of small tubules 25–30 nm in diameter. The appearance of morphologically mature interstitial cells of Leydig only adjacent to degenerated seminiferous lobules in the caudal portion of the testis suggests the possibility of local feedback control of Leydig-cell development.     

Ultrastructural alterations in hexafluoroacetone-induced testicular atrophy in the rat

Testicular atrophy was induced in rats by dermal application of hexafluoroacetone (HFA) at 39 or 130 mg/kg/day for 14 days, but not at a dosage of 13 mg/kg/day. Affected germ cells were mostly spermatids and to a much lesser extent spermatocytes; spermatogonia were unaffected. Late spermatids were retained in Sertoli cells and showed degenerative changes. Sertoli cells exhibited cytoplasmic vacuolation, distended endoplasmic reticulum, and a marked increase in lipid droplets. Leydig cells exhibited a slight increase in lipid droplets, fewer mitochondria, and diminution and segregation of the agranular endoplasmic reticulum from mitochondria. A correlation between ultrastructural and biochemical changes in HFA-induced testicular atrophy is presented.     

Ultrastructure of human Leydig cells at early gonadal embryogenesis

The ultrastructure of human Leydig cells at different stages of the testicular prenatal development is described by means of transmission and scanning electron microscopy. Between 5 and 7 weeks of gestation (w.g.) the interstitial tissue of the gonad is filled with small undifferentiated mesenchymal cells, migrating primordial germ cells and blood vessels. When the embryo is 7 to 8 weeks-old Leydig cells (LC) appear in basically two morphological patterns, light and dark cells. Their most significative feature is the development of the smooth endoplasmic reticulum (SER) as a dense tubulo-vesicular network and the presence of numerous pleomorphic mitochondria with mainly lamellar cristae. At 14 and 16 w.g. the testicular interstitium reaches the maximum development; the cytoplasm of the LC shows a widespread network of anastomosing tubules of the SER and mitochondria with tubular cristae. Fetal LC show a partial cell coat, lack the crystals of Reinke, have few lipid droplets and show no signs of massive cell degeneration, at least until 16 w.g. These ultrastructural modifications in fetal LC are in accordance with the changes in both steroidogenic activity and hCG levels reported by the literature to occur at this stage of development. Junctional complexes were often observed among LC from 7 to 8 w.g. onwards.     

Malignant Sertoli and Leydig cell tumour in a boar

A rare case of Sertoli and Leydig cell tumour was investigated in a 6-month-old boar. The neoplasm was found in the right testis and had metastasized to the liver, spleen, kidneys and diaphragmatic peritoneum. Metastatic nodules were also present in the tissues near the right testis and some neoplastic cells were present in the superficial inguinal lymph node. The neoplastic cells exhibiting severe pleomorphism were divided into Sertoli and Leydig cell types, although in some sites, it was not possible to classify tumour cells clearly as Sertoli or Leydig in type. In the metastatic lesions there were anaplastic Sertoli cells with abundant collagen fibres. Some neoplastic Sertoli cells and a few neoplastic Leydig cells revealed cytoplasmic reactivity for testosterone. Ultrastructurally, the neoplastic cells were characterized by mitochondria with tubulovesicular cristae, smooth endoplasmic reticulum, membranous structures, lysosomes, lipofuscin granules or lipid droplets.     

Morphological studies on the origin of adult-type Leydig cells in rat testis

The differentiation of adult-type Leydig cells (ATLC) in rat testis was studied with the help of electron microscopy and a combined technique of autoradiography and enzymehistochemistry. Male Wistar rats on each alternative day from postnatal day (pnd) 9 to 25 were perfused in situ, and the testicular sections were observed by electron microscopy. The cytoplasm of the ATLC contained smooth endoplasmic reticulum, mitochondria, a few small lipid droplets and a large well developed Golgi apparatus. In addition to typical fibroblasts/fibrocytes, a group of fibroblast-like cells was observed in the peritubular and perivascular spaces of the interstitial tissue. These cells, containing smooth endoplasmic reticulum and a large number of mitochondria with tubular-type cristae, were named as intermediate-type cells. In the autoradiographic experiments the 3-H-thymidine and 14-C-thymidine uptake behaviour of fetal-type Leydig cells, ATLC, intermediate-type cells and myoid cells was studied from 1 till 25 pnd. The intermediate-type cell, observed predominantly between pnd 9 to 21, showed the highest labelling index. The absolute number of fibroblasts/fibrocytes showed a peak on pnd 13 and decreased gradually with age. The duration of DNA synthesis phase ranged for interstitial fibroblasts: 7.9 h, for intermediate-type cell 10.4 h, for ATLC 17.4 h. The results lead to the conclusion that ATLC most probably originate to a large extent from the peritubular and to a lesser extent from perivascular fibroblast-like cells of the interstitial tissue.     

Hyperplasia and the immature appearance of Sertoli cells in primary testicular disorders

Testicular biopsy specimens from adult patients affected with cryptorchidism, Klinefelter's syndrome, and Del Castillo's syndrome were examined by light and electron microscopy. The study revealed a high proportion of testes showing seminiferous tubules with hyperplasia of Sertoli cells (from 25 to 45 cells per transverse tubular section). These cells had an immature appearance and showed a pseudostratified distribution. The nucleus was round to ovoid and regular in outline, with a smaller nucleolus than that of mature Sertoli cells. The cytoplasm showed less development of the endoplasmic reticulum as well as of the secondary lysosomes and lipid droplets than that in mature Sertoli cells. Characteristic features of these immature Sertoli cells were abundant cytoplasmic microfilaments, elaborate interdigitations between adjacent cells, and extensive tight junctions, from basement membrane to lumen. In the cryptorchid testes, a more immature Sertoli cell was found to constitute the majority of the cells in hypoplastic zones. In Klinefelter's and Del Castillo's syndromes as well as in cryptorchid testes to a lesser degree, a transitional type of cell-from immature to mature-was also observed. These observations suggest that Sertoli cells in these primary testicular disorders reflect a congenital deficiency producing abnormal development.     

The fine structure of the zona glomerulosa and the zona fasciculata of the adrenal cortex of the opossum

The normal fine structure of the zona glomerulosa and the zona fasciculata of the opossum is described, with emphasis on the structure of the smooth surfaced endoplasmic reticulum (SER) and mitochondria. The cells of the zona glomerulosa are characterized by the presence of numerous rod-shaped mitochondria with shelf-like cristae, small amounts of tubular SER and moderate amounts of rough surfaced endoplasmic reticulum. The cells of the zona fasciculata possess large quantities of SER, spherical mitochondria with tubular cristae and a few profiles of rough surfaced reticulum. A Golgi apparatus is present in cells of both zones as are coated vesicles, coated invaginations of the plasmalemma, lysosomes and lipid droplets. It is concluded that the tubular SER seen in electron micrographs corresponds closely to the form of this membrane system found in the living cell. The functional significance of the associations observed among lipid droplets, mitochondria and SER are discussed.     

Ultrastructure and immunohistochemistry of a fetal-type Leydig cell tumor.

A symptomless scrotal mass was removed from a 34-year-old man. The lesion was 7 cm in diameter and it was grossly a hemorrhagic cyst with indurated walls. By light microscopy tumor cell clusters and cords were seen infiltrating the testicle, tunica albuginea, and paratesticular tissue. In the immunohistochemical analysis the tumor cells were immunoreactive with anti-S-100 protein and anticarcinoembryonic antigen, but they did not express cytokeratin or alpha-fetoprotein as tested with paraffin sections. Tumor cell clusters were enveloped by a laminin-positive basement membrane. Electron microscopy revealed abundant smooth endoplasmic reticulum, lipid droplets, and membranous whorls in the cytoplasm. Lamellar whorled bodies were also seen in mitochondria, which contained tubulovesicular cristae. The presence of a well-developed, often multilayered basement membrane was confirmed at ultrastructural level. The activity of 3-beta-hydroxysteroid dehydrogenase suggested that the tumor cells were capable of androgen synthesis. The morphological features are reminiscent of fetal-type Leydig cells and are distinctly different from the Leydig cell tumors described so far.     

The fine structure of testicular interstitial cells in mice

The testicular interstitial cells of mice contain an abundant agranular endoplasmic reticulum occurring as a network of interconnected tubules. Unusual features of the reticulum are occasional extensive whorls of concentric membranes and the occurrence of bundles of parallel double-walled tubules. The mitochondria have tubular cristae and are occasionally very large. In contrast to the interstitial cells of other species that have been described, the mouse interstitial cells show a segregation of the cytoplasm into areas of dense agranular reticulum and other areas where the agranular reticulum is relatively sparse. The latter areas contain scattered cistenae of granular reticulum, many free ribosomes in clusters, mitochondria, lipid droplets and small granules. The mitochondria and lipid droplets are often encircled by cisternae of the reticulum. Biochemical evidence from other laboratories, taken with the present results, indicates that the membranes of the endoplasmic reticulum in mouse interstitial cells are the site of the enzymes that mediate the synthesis of testosterone from progesterone. There is also an indication that cholesterol biosynthesis is associated with the agranular reticulum. The membranes of the agranular reticulum may also serve as a reservoir for the storage of cholesterol.     

Liposarcoma. An ultrastructural study of 15 cases

Fifteen liposarcomas from 13 patients were examined by electron microscopy. These included nine primary tumors, four recurrent tumors after primary surgery or irradiation, and two metastatic lesions. Twelve of the liposarcomas were located in the thigh, and 11 were of the myxoid variety. All neoplasms were composed of cells having the ultrastructural characteristics of some stage of lipoblastic differentiation, i.e., lipid droplets, micropinocytotic vesicles, glycogen, external lamina, intermediate filaments, Golgi apparatuses, rough and smooth endoplasmic reticulum, and mitochondria. The nuclear-cytoplasmic ratio and nuclear pleomorphism were related inversely to the size and number of lipid droplets. Lipoblasts were frequently in close association with capillaries and pericytes, and in four cases lipid droplets were found in pericytes. Multivacuolated, mitochondria-rich lipoblasts, resembling brown fat cells, also were seen. Most tumors contained lipid-free, poorly differentiated mesenchymal cells that showed a continuum of morphologic differentiation to cells that closely resembled early lipoblasts that contained nonmembrane-bound lipid vacuoles. Fibrolipoblasts, cells containing lipid droplets and abundant rough endoplasmic reticulum, were observed only in well-differentiated liposarcomas. Some soft tissue sarcomas contain vacuolated cells that simulate lipoblasts by light microscopy but prove to be reactive or malignant fibroblasts, histiocytes, or smooth muscle cells ultrastructurally. Therefore, use of electron microscopy may be necessary to establish the line of differentiation in these neoplasms.     

Electron microscopy of a feminizing leydig cell tumor of the testis

The ultrastructural characteristics of a feminizing interstitial (Leydig) cell tumor of the testis were compared with those of normal Leydig cells and with the findings described in 10 published cases of Leydig cell tumor. The neoplastic Leydig cells superficially resembled normal Leydig cells. Similarities included abundant smooth endoplasmic reticulum, lipid, and microbodies. Contrastingly, Reinke crystalloids and paracrystalline inclusions were absent and lipochrome pigment and lysosomes very rare. The nuclei were large and contained enlarged, often multiple, nucleoli. The nuclear membranes tended to be irregular and undulating. Cytoplasmic membranous whorls and myelin figures were conspicuous. Fairly homogeneous fibrous septa were evident between single and grouped tumor cells. Despite several individual variations, there is a general resemblance between the neoplastic Leydig cells in this patient and those previously reported. No distinguishing ultrastructural characteristics were discerned between feminizing and virilizing Leydig cell tumors.     

In vivo differentiation of brown adipocytes in adult mice: An electron microscopic study

The differentiation of brown adipocyte precursor cells was studied in interscapular brown adipose tissue of adult mice by electron microscopy. Different stages of cell differentiation were characterized in situ. Previous autoradiographic studies suggested that interstitial cells represent the precursor cells of fully differentiated brown adipocytes. The present observations provide morphological evidence for a progressive differentiation of interstitial stem cells into mature brown adipocytes. Four typical stages of development were identified: (1) interstitial cells, (2) protoadipocytes, (3) preadipocytes, and (4) mature brown adipocytes. Interstitial stem cells were small spindle shaped cells, situated between brown adipocytes and characterized by a high nuclear-cytoplasmic ratio, the scarcity of organelles, and the absence of lipid inclusions. Protoadipocytes resembled interstitial cells except that they contained a few tiny lipid droplets in their cytoplasm. Preadipocytes had a larger cytoplasm enclosing many mitochondria and lipid droplets; the smooth endoplasmic reticulum was well developed surrounding the lipid droplets, and was closely associated with the mitochondria. Preadipocytes had the typical structure of growing cells, developing long cytoplasmic processes between and around blood capillaries. Mature brown adipocytes represented the final stage of differentiation. Almost all their cellular volume was occupied by lipid droplets and numerous mitochondria with very dense cristae. Brown adipocytes were also characterized by a tight association with blood capillaries, as expected from metabolically active cells requiring oxygen and substrates. These observations provide direct ultrastructural evidence for a progressive differentiation of interstitial cells into brown adipocytes with a continuum of intermediate cellular types.     

Androgen producing adrenocortical carcinoma

Summary Two cases of androgen secreting adrenocortical carcinoma have been described by light and electron microscopy. The histological and ultrastructural features of the tumour cells were similar to those of compact cells of zona reticularis and to those described in virilizing adenomas. They possess numerous mitochondria with lamellar and tubular cristae, abundant smooth endoplasmic reticulum, lipofuscin bodies and scanty lipid. Irregularly shaped, crenated mitochondria, with outpouchings of the outer limiting membrane have also been observed. The clusters of neoplastic cells were surrounded by basement membrane which demonstrated a focal discontinuity, probably reflecting malignancy of the tumours. Hyperplasia of smooth endoplasmic reticulum and the presence of outpouchings of the mitochondrial outer limiting membrane might be the morphological manifestation of endocrine activity of the tumours.     

Ultrastructural study of osteosarcomas

The ultrastructural examination of four osteosarcomas (osteogenic, undifferentiated, and pleomorphic) is described. There are three types of tumor cells. Most of the cells are held in contact by desmosome-like tight junctions; they are atypical osteoblasts with cytoplasmic processes, dilated rough endoplasmic reticulum, mitochondria carrying calcific inclusions, lipid droplets surrounded by glycogen, and intracellular fine filamentous fibers. Other cells exhibiting varying degrees of osteoblastic maturity are also seen with multilobed nuclei, a clear cytoplasm, and straight bordered membranes. The last type is chondroid with abundant deposits of glycogen, lipid droplets, and undilated rough endoplasmic reticulum. The matrix is composed of fibrils without periodicity, scattered and deteriorated collagen fibers, and focal calcium deposits of hydroxyapatite crystals as in embryonal bone, dentine, or callus bone.