The regenerative capacity of testicular interstitial tissue

Summary A single intraperitoneal injection of ethane dimethanesulphonate (EDS) destroys all Leydig cells in the adult rat testis but 1–2 weeks later new foetal-type Leydig cells begin to regenerate within the interstitial tissue. A further EDS treatment at 4 weeks failed to kill the new population of foetal-type Leydig cells. Between 10–20 weeks, the new Leydig cells exhibited the characteristics of adult-type Leydig cells. These cells responded to another EDS treatment by exhibiting a second phase of complete degeneration followed by regeneration of a foetal-type and subsequently an adult-type cell population. The results indicate that the testis retains the ability to replenish its supply of Leydig cells despite successive phases of total degradation of Leydig cells.     

Ultrastructural analysis of the effect of ethane dimethanesulphonate on the testis of the rat,guinea pig,hamster and mouse

Summary The morphological response of the testis of rats, guinea pigs, Syrian hamsters and mice to treatment with the cytotoxin ethane dimethanesulphonate was examined using light and electron microscopy. One to two days after a single administration of ethane dimethanesulphonate to adult rats, guinea pigs, and hamsters, the Leydig cells showed marked ultrastructural alterations suggestive of degeneration and cell death. The former alterations included karyopyknosis, cytoplasmic vesiculation and accumulation of lipid inclusions and large lipofuscin bodies. Fragments of necrotic Leydig cells were often engulfed by the interstitial tissue macrophages. The morphology of the seminiferous epithelium of these three species was unchanged from the morphology observed in vehicle-injected control animals. In contrast, multiple injections of ethane dimethanesulphonate given to mice produced no ultrastructural alterations to Leydig cells yet the seminiferous epithelium exhibited disruption of spermatogenesis. Although the Leydig cells of the mouse appear resistant to ethane dimethanesulphonate, this agent exerts a selective cytotoxic action upon Leydig cells of the rat, guinea pig and hamster thus identifying ethane dimethanesulphonate as a useful chemical for future endocrine and physiological studies of testicular function in three common laboratory species.     

Regeneration of Leydig cells in unilaterally cryptorchid rats: evidence for stimulation by local testicular factors

Summary Leydig cells in testes of adult rats were selectively destroyed by a single intraperitoneal injection of ethane dimethane sulphonate. Four days later rats were made unilaterally cryptorchid and 1, 2 and 4 weeks later the histology of the testes was examined by light microscopy and morphometry. After induction of unilateral cryptorchidism, the volume of abdominal compared to scrotal testes was reduced by 45–60% due to rapid impairment of spermatogenesis in abdominal testes. Leydig cells were not present in either scrotal or abdominal testes in the 1-week unilateral crytorchid group. A new generation of foetal-type Leydig cells was observed in scrotal testes of the 2-week unilateral crytorchid group although their total volume per testis estimated by morphometry, was small, being approximately 1 l. In contrast, the abdominal testis exhibited a remarkable proliferation of foetal-type Leydig cells (total volume per testis, 16 l) which predominantly surrounded the peritubular tissues of the seminiferous tubules. A similar morphology and pattern of Leydig cell development was observed in scrotal and abdominal testes of the 4-week unilateral cryptorchid group where total Leydig cell volume was 7 l vs 21 l, respectively. The results show that regeneration of a new population of Leydig cells occurs more rapidly in the abdominal testis than in the scrotal testis of the same animal. These observations suggest the possibility that augmentation of Leydig cell growth is mediated by local intratesticular stimulatory factors within the abdominal testis. Development of new Leydig cells from the peritubular tissue provides circumstantial evidence that the seminiferous tubules and in particular the Sertoli cells, are a likely source of agents that stimulate the growth of Leydig cells.     

Origin of regenerating Leydig cells in the testis of the adult rat

Summary Ethane dimethanesulphonate (EDS) was used as a specific cytotoxin to eliminate the Leydig cell population of the adult rat testis. Ultrastructural, morphometric and serum gonadotrophin and testosterone analysis was used to study the response of the intertubular tissue of the testis from 1 day to 10 weeks after EDS treatment. In control animals, the testis contained approximately 28 million Leydig cells and 8 million macrophages. Three to seven days after EDS treatment, Leydig cells were absent and serum testosterone was undetectable. Macrophage numbers increased three-fold by 3 days and returned to pretreatment values thereafter. At 2 and 3 weeks post-EDS, foetal-type Leydig cells (1–2 million per testis) appeared in proximity to perivascular and peritubular tissues, a feature also observed at 4 weeks when numerous such cells (15 million per testis) formed prominent clusters in perivascular and peritubular locations. Between 6 and 10 weeks after EDS treatment, the foetal-type Leydig cells were transformed morphologically into adult-type Leydig cells, they occupied central intertubular positions and their numbers were restored to pretreatment values. Regeneration of Leydig cells was reflected by elevated serum testosterone levels which returned towards the normal range. The results demonstrate the regenerative capacity of the testicular intertubular tissue and indicate a dual site of origin of Leydig cells which initially resemble foetal-type Leydig cells prior to establishing the adult-type Leydig cell population. The morphological pattern of Leydig cell regeneration suggests that in addition to gonadotrophic stimulation, local testicular factors from the seminiferous tubules may stimulate Leydig cell growth.     

Morphometric changes in the adrenal medulla of intact and ethane dimethanesulphonate (EDS)-injected rats subjected to chronic treatment with isoproterenol or propranolol

Morphometric characteristics of adrenal medulla were analysed stereologically in adult male rats injected with a single dose of ethane dimethanesulphonate (EDS), an agent that causes atrophy of the inner adrenocortical zone, or vehicle, and subjected to isoproterenol (ISO) or propranolol (PROP) treatment over the following 15 days. Compared with dimethylsulphoxide (DMSO) vehicle-injected controls, in EDS-administered rats the volume of chromaffin cell nuclei was decreased. ISO treatment in these rats increased the volumes of chromaffin cells and their nuclei. Furthermore, in both EDS- and vehicle-injected rats ISO significantly enlarged the total volume of medullary blood vessels, suggesting a vasodilatatory effect of β-adrenoceptor stimulation. However, unexpectedly, in EDS-injected rats PROP treatment also caused an increase in the volumes of chromaffin cells and their nuclei. This finding, most likely, may be related to a non-β-adrenoceptor-related action of PROP. Collectively, the present results suggest that the response of adrenomedullary chromaffin cells to chronic ISO-induced β-adrenoceptor stimulation is dependent on the functional status of adrenal cortex, so that the stereologically detectable changes were found only in rats previously exposed to the destructive action of EDS on the adrenal gland cortical cells. Moreover, they indicate that ISO treatment exerts a reversing effect on the morphometrical changes of chromaffine cells induced by EDS administration.     

Selective destruction and regeneration of rat Leydig cells in vivo

Summary The effect of a single i.p. administration of ethane dimethanesulphonate (EDS) upon rat testicular histology was studied by light microscopy and morphometry up to 4 weeks after treatment. One day after injection the interstitial tissue exhibited degenerating Leydig cells, abundant pyknotic interstitial cells, deposition of cellular debris and extensive networks of fibrillar material. Macrophages contained greatly increased numbers of cytoplasmic inclusion bodies. From 3 to 7 days morphometric analysis showed that Leydig cells and cellular debris had disappeared from the interstitial tissue, leaving only macrophages, fibroblasts and lymphatic endothelial tissue. A very small number of new Leydig cells were seen on day 14, often located in peritubular or perivascular positions. Regeneration of foetal-like Leydig cells occurred by 4 weeks, their cytoplasm containing large lipid inclusions and, numerous Leydig cells were often observed closely applied to the walls of the seminiferous tubules. The observations suggest that, after experimental destruction and depletion of Leydig cells, an interstitial precursor cell, as yet unidentified, gives rise to a new Leydig cell population. EDS thus offers a valuable opportunity to study further the interactions between the seminiferous tubules and the interstitial tissue following the destruction and subsequent regeneration of the Leydig cells.     

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats

Summary Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6–10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal Leydig cells resulting in their disappearance by day 7. Destruction of foetal Leydig cells was followed by an arrest of testicular growth in comparison to testes of intact age-matched control rats. In testes of EDS-treated rats, gonocytes and spermatogonia also degenerated, forming pyknotic bodies within the seminiferous cords. In contrast, interstitial fibroblasts and mesenchymal cells showed proliferative activity, which on days 4 and 5 after treatment resulted in peritubular hyperplasia surrounding each seminiferous cord. Thereafter, on day 21 after EDS administration, the previously depressed serum testosterone levels became markedly elevated coincident with the development of many immature-type Leydig cells, of which the total volume per testis was similar to that of Leydig cells in control testes, despite a four- to five-fold difference in testicular volumes. The results indicate that, although EDS destroys the foetal Leydig cells and impairs spermatogenesis, the interstitial tissue exhibits increased cell growth. The latter probably occurs in response to altered gonadotrophic stimulation and/or disturbances in the interaction between the seminiferous cords and the interstitial tissue.     

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augumentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 m³ to 637 m³, but the number of Leydig cells in paired testes increased from 53x10⁶ to 113x10⁶. The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.Visiting scientist to Laboratory of Electron Microscopy (Director: Prof. L.J. Pelliniemi)     

Response of the seminiferous epithelium of the rat testis to withdrawal of androgen: evidence for direct effect upon intercellular spaces associated with Sertoli cell junctional complexes

The morphological response of the Sertoli cells to partial or complete withdrawal of testosterone was studied in adult rats following hypophysectomy or administration of ethane dimethanesulphonate (EDS), a toxicant known to destroy selectively the Leydig cells of the testis. To assess the role of germ cells in effecting changes to Sertoli cells following withdrawal of testosterone, germ cell-deficient rats with Sertoli-cell-only testes (SCO) were treated with EDS to remove the source of testosterone. At 6 days after hypophysectomy or 4,6 and 8 days after EDS treatment, stage VII and VIII seminiferous tubules showed degenerating germ cells and numerous basally-located vacuoles approximately 1–15 m in diameter. Ultrastructural analysis indicated that most of the vacuoles were multiple focal dilations of the intercellular space associated with Sertoli cell junctional complexes. In SCO rats, treatment with EDS resulted in a significant (P<0.05) increase in the formation of many vacuoles particularly in the base but also in the trunk of the Sertoli cells and again electron microscopic analysis showed multiple, localized expansions of the intercellular space associated with Sertoli cell junctional complexes. The appearance of intercellular spaces in SCO testes following androgen withdrawal cannot be attributed to shrinkage of degenerating germ cells since the seminiferous tubules did not contain germ cells. It is concluded that withdrawal of androgen induces early morphological alterations of the Sertoli cell junctional complexes in which the sites of membrane fusions representing tight junctions remain intact whereas the intercellular spaces exhibit major focal dilations. The results are discussed in relation to the fluid secretion by the seminiferous tubules which is regulated by the Sertoli cells.     

Macrophages in the interstitial tissue of the rat testis

Summary Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12–14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.     

Innervation of the testicular interstitial tissue in reptiles

Summary In the tortoise Testudo graeca, the lizards Lacerta dugesi and Lacerta pityusensis, and the snake Natrix natrix, the innervation of the testicular interstitial tissue was studied by light and electron microscopy, the acetylcholinesterase (ache) technique, the Falck-Hillarp method for the detection of catecholamines, and the application of 6-hydroxydopamine. The intertubular spaces of the reptilian testes studied contain adrenergic nerve fibers the amount and distribution of which varies considerably both in various species and in various stages of the reproduction cycle. Nerve fibers do not enter the seminiferous epithelium. Fluorescence microscopy of the lizard testis reveals catecholaminergic varicosities which are mainly arranged around blood vessels, but do not show obvious connexions to Leydig cells. Ache-positive fibers are equally distributed in lizard testes surrounding each seminiferous tubule. In Natrix natrix ache-positive fibers are irregularly spread among groups of tubules, without showing a definite relation to Leydig cells either. By electron microscopy bundles of unmyelinated axons and axon terminals can be more easily detected in the testes of immature animals than in adult. Terminals of nerve fibers containing small (400–500 Å in diameter) and large (800–1400 Å) dense-cored vesicles and sometimes small clear vesicles establish contacts with Leydig cells. Three types of contact are described. 1. Contacts par distance at a distance of about 2000 Å and basal lamina interposed; 2. membranous contacts having a 200 Å gap only between axolemma and Leydig cell plasmalemma; 3. invaginations of terminals into Leydig cell perikarya. The latter may exhibit surface specialisations, which strongly resemble postsynaptic membrane thickenings. Experiments using 6-hydroxydopamine underline the adrenergic character of testicular nerve fibers, which can be regarded as another example of non-cholinergic, ache-positive neurons. In the testis of the immature tortoise profiles of axons occur which probably represent purinergic, ache-positive neurons.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1).I am much indebted to Mrs. R. Sprang for her skillfull technical assistance.     

Organization of testicular interstitial tissue of an Australian rodent,the spinifex hopping mouse,Notomys alexis

Summary The organization of testicular interstitial tissue of the spinifex hopping mouse, Notomys alexis differs from that of other rodents. It comprises between 10.3% and 17.3% (average 15.0%) of the total testicular volume, and is variable in its organization both at different locations within the testis of the one animal and among different individuals. Abundant, closely packed Leydig cells are usually present; however, in some regions large, thick-walled blood vessels and extensive peritubular lymphatic spaces, often lacking an endothelium adjacent to the Leydig cells, are also prominent. The Leydig cells in contact with the large blood vessels and lymphatics, unlike those in regions where lymph is sparse, are not densely packed and sometimes contain numerous lipid droplets. Ultrastructure of Leydig cells is typical of steroid-producing cells; however, mitochondria are often extremely large, unusual in shape or bizarely arranged in relation to one another. Also electrondense bodies displaying a paracrystalline-like internal structure of parallel, electron-dense filaments arranged in a lattice pattern occur in the cytoplasm of many cells. The significance of these unusual ultrastructural features and the organization of the interstitial tissue remain to be determined conclusively, but may relate to steroid synthesis, secretion and uptake.     

Changes in the fine structure of the testicular Leydig cells of the seasonally-breeding bat,Myotis adversus

Summary Leydig cells of the bat, Myotis adversus, have been examined by electron microscopy throughout fourteen months. During the breeding season the Leydig cells become hypertrophied and are characterised by prominent areas of agranular endoplasmic reticulum and numerous small, membrane-bound granules. Microperoxisomes are also observed. During the period of testicular regression. Leydig cell size and the number of membrane-bound granules are greatly reduced. Lipid droplets and dense bodies are more numerous.     

Seasonal changes in the testicular morphology and interstitial tissue histomorphometry of Sahraoui camel under Algerian extreme arid conditions

The seasonal testicular morphology and the morphometry of the interstitial tissue were studied in 62 camels at Algerian extreme arid region. The maximal testicular size was recorded during the rutting season. In this period, the interstitial tissue occupied high area and volume with significant increase of the intertubular constituent’s volume, hypertrophy of the Leydig cell, and maximal number of Leydig cells per testes. Therefore, the highest ratios of seminiferous tubules to interstitial tissue area and volume and the highest fraction of intertubular empty space were recorded during the non-rutting. The greater Leydig cell nucleus size was observed during the post-rutting season. Finally, the numerical density of Leydig cells did not significantly change over the year. These results provide information on the relationship between seasonal changes of camel testicular morphology and the histomorphometry of the testicular endocrine compartment in camels at the arid livestock conditions of the southeastern Algerian desert.     

A morphological study on Leydig cells of scrotal hyperthermia applied rats in short-term

Testicular function is highly dependent on temperature control. The aim of this study was designed to investigate the morphological changes and regulation of steroidogenesis by light and electron microscopic level in Leydig cells (LC) after scrotal hyperthermia in rats. The rats were randomly allotted into one of four experimental groups: A (Control), B (1 day after scrotal hyperthermia), C (14 days after scrotal hyperthermia), D (35 days after scrotal hyperthermia); each group contain seven animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for 6 consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia applied rats were sacrificed under 50 mg/kg ketamine anaesthesia after 1, 14 and 35 days, and biopsy materials of testis were obtained for light and electron microscopic examinations. To date, no histopathological changes of LC injury after scrotal hyperthermia in rats have been reported. Light microscopic examinations indicated increase degenerative LC, decrease in number of testosterone positive LC in interstitial area after scrotal hyperthermia in short-term. In scrotal hyperthermia, a dilated smooth endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some LC displayed deep invaginations and irregular outlines. The number of lipid droplets was very considerably increased in most LC when compared to control group. As a conclusion, we claim that temperatures higher than the body temperature may cause infertility by damaging LC.     

Effects of scrotal hyperthermia on Leydig cells in long-term: a histological,immunohistochemical and ultrastructural study in rats

We have previously demonstrated that scrotal hyperthermia induce Leydig cell (LC) damage in short-term. The objectives of this pilot study were to investigate morphological changes and regulation of steroidogenesis on LC in long-term and the time of observation were extended to investigate whether the LC would eventually make a recovery after scrotal hyperthermia. The rats were randomly allotted into one of four groups: A (control), B (70 days after scrotal hyperthermia), C (105 days after scrotal hyperthermia), D (140 days after scrotal hyperthermia); each group contain seven animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for six consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia applied rats were sacrificed under 50 mg/kg ketamine anaesthesia after 70, 105 and 140 days, and biopsy materials of testes were obtained for light and electron microscopic examinations. Morphologically normal and the number of testosterone positive LC was significantly higher in 140 days after last heat than all other heat treatment groups. In heat treated groups, a dilated smooth endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. In the 140 days after scrotal hyperthermia, the severities of degenerative changes of LC were less than that observed in the other heat treated groups. We conclude that, scrotal hyperthermia cause morphological damaging and impaired steroidogenesis in LC and recovery of these findings were noted first time in 140 days after the last heat treatment.     

Role of testicular interstitial macrophages in regulating testosterone release in hyperprolactinemia

Hyperprolactinemia-induced hypogonadism has been linked to a dysfunction of the hypothalamus-pituitary-testis axis. The direct inhibitory effects of prolactin on the testicular release of testosterone have also been demonstrated, though their mechanisms remain unclear. Incubation of rat testicular interstitial cells (TICs) with prolactin stimulated the release of testosterone. TICs from rats with anterior pituitary-grafting-induced hyperprolactinemia release lower amounts of testosterone than controls. However, Leydig cells isolated from anterior pituitary-grafted rats release a greater amount of testosterone. These paradoxical observations have remained unexplained. This study examined the roles of testicular interstitial macrophages and of their product, tumor necrosis factor-alpha (TNF-alpha), in regulating Leydig cells under condition of hyperprolactinemia. Hyperprolactinemia was induced by grafting two anterior pituitary glands of rats under the renal capsule. Control animals were grafted with rat cortex tissue. The rats were sacrificed 6 weeks later. TICs and macrophages, and Leydig cells were isolated for in vitro incubation and drugs challenge. Testosterone released by testicular interstitial or Leydig cells was measured by radioimmunoassay. TNF-alpha concentration in the medium of TICs or macrophages was measured by enzyme-linked immunosorbent assay (ELISA). A dose-dependent stimulation of TNF-alpha secretion in the medium of TICs or macrophages by the prolactin challenge was observed. Higher amounts of TNF-alpha were released by TICs in the anterior pituitary-grafted rats than in the control group. In contrast, the release of TNF-alpha by testicular interstitial macrophages isolated from the anterior pituitary- and cortex-grafted groups was quantitatively similar. Challenge with human chorionic gonadotropin did not modify the TNF-alpha release by testicular interstitial macrophages in either group. Challenge of Leydig cells with TNF-alpha inhibited their release of testosterone stimulated by human chorionic gonadotropin, but not their basal testosterone release. These different patterns of testosterone release in TICs versus Leydig cells cultures in anterior pituitary-grafted rats may be due to the influence of testicular interstitial macrophages. These observations correlate with in vivo conditions, where prolactin increases the release of TNF-alpha by testicular interstitial macrophages, which, in turn, decreases the human chorionic gonadotropin-stimulated release of testosterone by Leydig cells. In summary, hyperprolactinemia-induced hypogonadism involves a mechanism of prolactin-originated, macrophage-mediated inhibitory regulation of testosterone release by Leydig cells. TNF-alpha, one of the cytokines secreted by macrophages, may play a key role in this mechanism.     

Crystalloid formation in Leydig cells of rats (Rattus fuscipes)

Summary The ultrastructure of Leydig cells in a seasonally breeding rodent, Rattus fuscipes, was studied in the breeding and non-breeding season and compared with Leydig cell morphology after suppression of gonadotrophin secretion induced by hypophysectomy or chronic administration of testosterone. Serum luteinizing hormone (LH) and testosterone (T) were measured and in-vitro T production by testes was assessed by stimulation with human chorionic gonadotrophin (hCG). In non-breeding wild-trapped rats and rats with experimental suppression of gonadotrophins, the Leydig cells were atrophied and exhibited variable amounts of cytoplasmic lipid and crystalloid inclusions, the latter commonly dominating the cytoplasmic area. Compared with fertile rats, serum LH and hCG-stimulated T production of experimentally regressed rats was significantly reduced, confirming structural features indicative of Leydig cell inactivity. Atrophy of Leydig cell nuclei was accompanied by the formation of unusual intranuclear vesicles sometimes containing small crystalloids. Ultrastructural analysis suggested transfer of the vesicles to the cytoplasm where their unification gave rise to much larger crystalloid bodies. Crystalloids occurred when serum LH was depressed and with either full (T treatment) or arrested spermatogenesis (hypophysectomy) suggesting that their formation is governed by pituitary function and is not dependent upon the degree of spermatogenic activity.     

Studies of the fine structure of ovarian interstitial tissue

Summary The fine structure of the thecal gland of the domestic fowl, is described for the first time. In the fowl, the glands are located as islets of lipid-laden cells in the theca interna and also in the interfollicular regions. They appear as well defined structures, organized like endocrine glands, quite different from the surrounding theca interna cells. Each gland is composed of two different cell types, the steroid-producing cell, and a cell type never described before, named the enclosing cell. Both cell types are surrounded by a common, distinct basal membrane. The steroid-producing cells are characterized by their content of organelles typical of steroid-producing cells in other organs. The enclosing cells are char cterized by their peripheral location within the gland and their membranous contact with the steroid-producing cells, long processes with desmosomes and their relation to the nerve fibers. They do not contain the organelles typically found in steroid-producing cells. So far, the real function of the enclosing cells is unknown. The following structures are demonstrated in ovarian steroid-producing cells of the fowl for the first time: cytoplasmic microtubules and filaments, intramatrical lipid-like droplets, attachment devices, the polarity of the steroidproducing cells of the thecal gland.