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The ultrastructure of the seminiferous tubules was studied in rats that had been subjected to whole body irradiation on the 19th day of gestation. The seminiferous tubules from 3 months-old irradiated animals are devoid of germ cells and contain only Sertoli cells. Compared with controls of the same age, the seminiferous tubule basal membrane is thickened and multilayered and several alterations are observed in the Sertoli cells. The most characteristic of these alterations are: (a) an abnormal number of nuclear heterochromatin clumps, (b) the presence of numerous cytoplasmic vacuoles and various sized lipid droplets, (c) elaborate interdigitations and junctions between adjacent cells, and (d) the presence of anomalous ectoplasmic specializations disposed perpendicularly to the Sertoli cell membrane.

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as cryptorchidism, fetal irradiation, and ligation of the ductuli efferents lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.

Sertoli cells have various functions: mechanical (creation of two compartments in the seminiferous tubules, migration of germinal cells), secretory (secretion of anti-Müllerian hormone, inhibin, androgen-binding-protein and estrogen) and phagocytic. We report an ultrastructural study of the rat Sertoli cell during maturation and consider possible correlations between the acquisition of certain morphological characteristics and certain functions. During fetal life, the Sertoli cell possesses differentiated zones of junction with the gonocytes and seems to have a role in the migration of the gonocytes towards the periphery of the seminiferous tubule. The Sertoli cell performs the phagocytosis of the gonocytes which degenerate during their migration, and seems to be the site of production of protein granules, whose presence can be related to the synthesis of anti-Müllerian hormone. After birth and before puberty, when the inclusions resembling secretory granules disappear, the Sertoli cell membranes in contact with spermatocytes II and spermatids differentiate, forming, through the differentiated junctional complexes, two compartments (adluminal and luminal) in the seminiferous tubules. Finally, they acquire the characteristics of active secretory cells, capable, in particular, of steroid synthesis.

The initial stages of the development of the seminiferous cords involve the differentiation and the aggregation of primordial Sertoli cells opposite to cells which acquire a mesenchymal-like aspect. The hypothesis that the development of the seminiferous cords depends on epithelial-mesenchymal relations between the two cell types was submitted to experimental test. Male gonadal primordia of rat fetuses were cultured in vitro in a synthetic medium containing the proline competitor, L-Azetidine-2-Carboxylic Acid. This drug is known to disturb the synthesis and secretion of collagen and proline-containing proteins. It prevents testicular organogenesis or destroys it if it has begun. It suppresses the expression of laminin and fibronectin in the gonadal primordium. These observations are taken as evidence that cellular correlations of the epithelial-mesenchymal type play a role in the development of the testis as they do in that of other organs.

In an evaluation of the effect of ethinyl estradiol (EE) on the differentiation of fetal mouse testes, the ratio of the seminiferous tubular region to the testicular tissue region, the ratio of Sertoli cells to gonocytes in tubule cross sections, and the size of Leydig cells were determined by the Texture Analyse System (T.A.S., Leitz) in histological preparations of the testes. The testes were those of fetuses taken from dams given orally 0, 0.02, 0.2 or 2.0 mg/kg of body weight of EE in olive oil from day 11 through day 17 of gestation and killed at term. From experimental and the control testes, five sections were taken at 40-micron intervals. The areas of the seminiferous tubular region and the testicular region were determined and the Sertoli cells and gonocytes in tubule cross section were counted in each of the five sections. The diameters of 100 Leydig cells selected at random were averaged. These data were analyzed by Student's t test. The seminiferous tubular region was significantly increased in the testes treated with 0.02 mg/kg of EE and significantly decreased in those treated with 0.2 mg/kg of EE. The number of gonocytes per tubule cross section was significantly increased in the testes treated with 0.02 or 2.0 mg/kg of EE. The number of Sertoli cells per tubule cross section and the number of Sertoli cells per gonocyte were significantly decreased in the experimental testes. The size of the Leydig cells was significantly decreased in the testes treated with 0.2 mg/kg of EE. These findings suggest that prenatal exposure to EE before testicular differentiation affects tubular formation, the proliferation of fetal Sertoli cells, and Leydig cell differentiation, resulting in disturbances of spermatogenesis.

The foetal testis of the field rat (Millardia meltada) shows seminiferous tubules and interstitium consisting of mesenchymal cells and differentiated Leydig cells associated with blood vessels. The tubules during the prenatal period show gonocytes and Sertoli cells. After birth, their diameter decreases but again increases progressively after postnatal days 9 and 10. Spermatogonia appear among the gonocytes on postnatal day 1 and primary spermatocytes on day 8. Secondary spermatocytes and spermatids are not seen up-to-day 23. Several hypertrophied Leydig cells are seen in the foetal testis on day 17 but are greatly increased in number on days 18 and 19. A few hours after birth the Leydig cells show a rapid decrease in their number. These fluctuations in the Leydig cells of prenatal and neonatal testes have been correlated to the rise and fall in testosterone production during these periods. The Leydig cells show the histochemical characteristics of actively steroid-secreting cells, which consist of the presence of diffuse lipoproteins and a few lipid granules consisting of phospholipids; no cholesterol and/or its esters could be demonstrated. Such lipids are not present in the cytoplasm of undifferentiated mesenchymal cells. The seminiferous tubules do not show any appreciable development of lipid changes.

Intra-abdominal cryptorchidism and infertility are universal features of the prune belly syndrome but they usually are not seen with posterior urethral valves. To understand further the differences between these patients we reviewed testicular histology in 5 fetuses from each of 3 groups: prune belly syndrome, posterior urethral valves and normals. Spermatogonia were noted in all but the numbers were significantly reduced in those with the prune belly syndrome and posterior urethral valves compared to normals: 8.6 and 13.4 versus 31.2 per 10 high power fields, respectively. The fetuses with the prune belly syndrome also had marked Leydig cell hyperplasia in contrast to the other groups. These findings suggest that the testicular pathological condition in patients with the prune belly syndrome is caused by more than mechanical cryptorchidism.

The course of divergent differentiation of coelomic epithelium in the indifferent gonad has been traced up to the stage when morphological sex signs are distinctly seen. Eleven human embryos fixed in Carnoy and Bouin's fluid have been studied from 6 to 10 weeks of gestation. Paraffin sections were stained with hematoxylin, ferric hematoxylin after Heidenhain. Basal membrane fibres were revealed by silver impregnation after Karupu. PAS-reaction after McManus and additional staining with hematoxylin and light green were applied, as well as the reaction for total proteins with bromphenol blue after Miguel--Calvo. Sex differentiation of coelomic epithelium in the gonad developing according to the male type begins a little later than in the ovary. In the epithelium of the forming testis a great amount of mitotic figures is observed that might be connected with the presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing presence of Y-chromosome stimulating cells for extra mitotic cycles. The increasing number of mitotic figures in the epithelium should be considered as a sign demonstrating that differentiation according to the male type has started. Superficial epithelium of the embryonic ovary has a peculiar false pseudostratified structure. This secures cellular reserve for the ovarian cortex formation from the external epithelium. The apical surface of the cells in the external epithelium has a border which is evidently formed by microvilli and revealed by PAS-reaction and bromphenol blue. Ovarian follicular cells and Sertoli's cells (sustentocytes) in testis have the common origin in the human fetus--from coelomic epithelium of the gonad germ.

The postnatal development of the Sertoli cell barrier, tubular lumen, fluid flow, and cytoskeletal elements in Sertoli and myoid cells was investigated in the Sprague-Dawley rat. With the aid of hypertonic fixatives, a barrier to the rapid entry of fluid was noted in the majority of tubules on the 15th and 16th postnatal (p.n.) days and was completely formed in all tubules prior to p.n. day 18. The actin forming the ectoplasmic specialization (ES), a cytoskeletal complex related to the occluding junctions composing the barrier, began its development during the period of initial barrier formation (16 p.n. day) and progressively attained its adult prominence. The ES developed its characteristic adult pattern and adult fluorescent intensity at about p.n. day 22. Some seminiferous tubules showed very small lumina as early as p.n. day 10. All tubules were not open until p.n. day 30. The size (diameter) of the lumen increased slowly from p.n. day 10 until p.n. day 30 when it started to increase rapidly until about p.n. day 50. Fluid flow in seminiferous tubules was detected as early as p.n. day 20 and increased in amount thereafter. Myoid cell actin filament bundles, running in parallel, were present at p.n. day 10. Actin formed a meshwork pattern characteristic of the adult on, or slightly prior to, p.n. day 22. These data indicate that there is a temporal relationship between the development of the actin cytoskeleton within the Sertoli cell and initial formation of the Sertoli cell barrier. Similarly, there is a temporal relationship between the development of the actin cytoskeleton of myoid cells and tubular fluid flow. The rapid increase in tubular lumen diameter, however, does not correlate with the initial development of Sertoli and myoid cytoskeletal elements.

A study has been carried out on the width of the seminiferous tubules, and the proportion of pre-germinal cells, in 174 stillborn children between 24 and 40 weeks of gestation, and in 146 children dying between the ages of birth and six years. There was a general diminution in the diameter of the seminiferous tubule throughout the last trimester and immediately before birth, followed by a slow and steady increase in the diameter of the tubule throughout childhood. The germinal cells within the seminiferous tubules appear to be under some slightly increased stimulatory influence during the last few weeks prior to delivery, and under considerable stimulation in the first 4 or 5 weeks after birth. Following this these cells appear to diminish in size and become hidden among the Sertoli cells, to be re-stimulated around the time of puberty.

The paper is devoted to the study of the very first stages of testicular organogenesis in the rat, i.e. to the study of the initial stages of formation of the seminiferous cords. It takes over and extends the results of a previous histological study (Jost, 1972). The tissues were fixed with glutaraldehyde or glutaraldehyde-paraformaldehyde and post-fixed with osmium tetroxide. Examination with the optical microscope of semi-thick sections confirms that the differentiation begins at the stage of 13 days after fertilization; an increasing number of primitive Sertoli cells appear, first in the depth of the gonad, join each other and form the seminiferous cords. Study with the electron microscope reveals three characteristic criteria of the early differentiation of the Sertoli cells, namely : 1) an abundant cytoplasm, only slightly dense to electrons and containing short laminae or vesicles of rough endoplasmic reticulum; 2) complex contact zones involving infoldings and interdigitations of the membranes; 3) a layer of microfilaments beneath the flattened surface of the cells, that limits the outer surface of the seminiferous cords. These differentiation processes taking place at the stage of 13 days, are described.

Single fetal (9th day) treatment with either diethylstilbestrol (DES) or allylestrenol (AE) caused a considerable decrease, both at the age of five days and six weeks, in the weight of the testicles and the diameter of the seminiferous cords, while the ratio of spermatogonia to primary spermatocytes increased. When measured either at the age of five days or six weeks, gonadotropin treatment [a combination of follicle stimulating hormone (FSH) and luteinizing hormone (LH)], administered twice daily for three days after the hatching, led to an increase in the above-mentioned parameter and to a shift in the cell ratio towards the control value. However, the absolute value of the controls treated with FSH-LH was by far not reached. The effect of perinatal treatment could be detected even in adulthood, namely, at the age of five days the response capability was relatively weak in the cockerels treated with DES and AE, while high responsiveness was observed at the age of six weeks. In some cases the relative value of the increment exceeded even that of the control; however in absolute term it was well below the control. On the basis of these experiments it might be concluded that hormonal imprinting evoked by FSH-LH treatment also occurs in the gonad damaged by DES and AE. The setting in of imprinting ameliorates the damages caused by DES and AE and increases the response capability of the cells.

15th day chick embryo testes, were cultivated in organ and tissue culture for 5 days, in semisynthetic media. The histological examination of the cultured testes shows a noticeable retardation in development compared with normal testes at the same age. Microscopic examination of cultured testicular cells, shows two types of cells, epithelial and fibroblasts. The most remarkable result, the absence of germ cells from the tissue culture.