Contractility and structure of adult rat seminiferous tubules in organ culture

Summary Isolated pieces of seminiferous tubules of adult rats were grown in organ culture for up to 8 weeks in Petri dishes on the surface of nutrient agar. The medium consisted of newborn calf serum, Eagle's minimum essential medium, glutamate and antibiotics. This method allowed observation of the contractions of the seminiferous tubules in the culture. Contractility, light and electron microscopic structure and histochemically demonstrable activities of alkaline phosphatase and ATPase of the tubule walls were studied at 1-week intervals. The contractility and alkaline phosphatase activity were maintained in the tubule wall for 3 weeks, and the activity of ATPase was maintained for 5 weeks. The thin filaments of the myoid cells, which are responsible for the contractility, were seen with the electron microscope in tubules cultured for 5 weeks. The organ culture method described in the present paper seems to be valuable for studies concerning the functioning of the myoid cells of the seminiferous tubules and the possibility that this is regulated by hormones.     

Short-term effect of androgen deprivation on intraluminal pressure and contractility of the rat epididymis

Short-term effects of bilateral castration, cyproterone acetate and unilateral efferent duct ligation on intraluminal pressures and spontaneous contractions in different regions of the epididymis were studied in the rat. Ligation of the efferent ducts for 5 days did not alter pressures or spontaneous contractions in any region of the epididymis. However, bilateral castration produced time-dependent changes in pressures and contractions in different segments. In the caput, the amplitude, but not the basal pressure or the frequency, of spontaneous contractions increased by Day 1 after operation. In the corpus, increments in the basal pressure and the amplitude of contractions occurred by Day 5 whilst the frequency of contractions was not changed. Similar effects were observed in the cauda by 3 days after castration. Changes in all regions of the epididymis were also mimicked by cyproterone acetate treatment (10 mg/rat per day, s.c. for 21 days). In addition, this drug increased the amplitude of contractions in the cauda. The effect of castration was abolished by testosterone propionate (0.2 mg/kg per day, i.m. for 5 days). The results support the suggestion that an enhancement of sperm transport through the rat epididymis occurs shortly after castration. The results also suggest that, in normal rats, androgens suppress the contractility of the epididymal tubule to ensure an optimal rate of sperm transport.     

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.     

Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue

We have studied the distribution of histochemically detectable alkaline phosphatase in cultures of seminiferous tubule fragments and of peritubular cells from prepubertal rats. The same material also was immunohistochemically evaluated for the presence of desmin-containing intermediate filaments. The comparative analysis of alkaline phosphatase and desmin positivity shows that alkaline phosphatase histochemistry selectively detects desmin-containing contractile cells in tubular and peritubular cell cultures. We propose alkaline phosphatase as a novel marker for myoid cells that can be of help in screening, defining, and eventually standardizing the exact composition of peritubular cell cultures, a model that is of increasing interest in the study of cellular interactions in the testis.     

Organ culture of human seminiferous tubules: A useful tool to study the role of nerve growth factor in the testis

Summary Seminiferous tubules from human testes were mechanically isolated, the cut edges were sealed, and the tubules were cultured in medium free of fetal calf serum (FCS). Degeneration of germ cells occurred during the culture period and was paralleled by a disruption of the seminiferous epithelium, a disturbance in morphology and function of Sertoli cells, and a thickening of the lamina propria. However, when tubules were cultured for 5 days in the presence of FCS, degeneration of the spermatogenic tissue was reduced. FCS increased the mitotic activity of germ cells, but did not maintain normal morphology and function of Sertoli cells and cellular elements of the lamina propria. The thickening of the tubular wall concurred with a change in phenotype of lamina-propria cells from myoid to fibroblastic. Addition of nerve growth factor (NGF) to the culture medium (i) maintained the myoid phenotype of lamina-propria cells, (ii) prevented thickening of the tubular wall, and (iii) stabilized Sertoli cell morphology and function. The effects of NGF appeared to depend on the trophic effects of FCS, since NGF alone had no influence on the maintenance of a regular morphology of the spermatogenic epithelium. The present results indicate a decisive role for NGF in stabilizing specific functions of seminiferous tubules.     

Functional activity of rat testicular cells in culture

Testicular cells from adult hypophysectomized rats were cultured for 10 or 12 days, and the effect of treatment with hCG (10 ng/ml) on testosterone and progesterone production and the activity of the Leydig cell enzyme, 3 beta-hydroxysteroid dehydrogenase, were studied. Regardless of hormone treatment, on 4th day in culture a decline in the steroidogenic activity of cultured cells could be observed. Treatment with hCG resulted in stimulation of steroidogenesis on days 6 to 10 in culture, as measured by testosterone and progesterone production. Hormone treatment stimulated or inhibited the enzyme activity depending on the presence or absence in the culture medium of 10(-6) M spironolactone, an inhibitor of 17 alpha-hydroxylase, or an anti-androgen, cyproterone acetate.     

Vitamin A modulation of basement membrane production by purified testicular myoid cells.

Purified myoid cells, isolated from prepubertal rat testes, cultured in a chemically defined medium for up to 1 week do not change their metabolic activities, evaluated as protein synthesis and secretion, during the culture time. We report that fibronectin, collagen IV, and laminin are synthesized and secreted by myoid cells as demonstrated by immunocytochemical and biochemical methods. The deposition of all three proteins was spotty, with different regional localizations. The effect of vitamin A on the synthesis and the secretion of the basement membrane components was also evaluated. Retinol supplementation induces a higher synthesis of fibronectin and laminin, whereas it does not change collagen IV synthesis and secretion. The secretion of the other two molecules is differentially regulated by retinol; in fact fibronectin secretion is increased, whereas laminin secretion is reduced. Similar results were obtained utilizing retinoic acid. The data we report in this paper show, for the first time, that purified testicular myoid cells synthesize and secrete fibronectin, collagen IV, and laminin and that synthesis and secretion of these components of the basement membrane are regulated by retinol. These findings reveal a new effect of vitamin A in the regulation of mammalian spermatogenesis.     

Postnatal development of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow

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.     

Culture of rat Sertoli cells isolated with a modified procedure. Morphological identification of cell population and cell reactivity

The effects of growing of Sertoli cells isolated from rat seminiferous epithelium by a modified procedure, and responses of these cells to dBcAMP or FSH stimulation was estimated using morphological methods. The modified isolation procedure included repeated mechanical rinsing of tubule pieces with a modified EDTA containing Hanks medium. Moreover, streptornase instead DNase was added to the trypsine containing medium and this resulted in a better dispersion of tubule components. The culture conditions remained unmodified. A high degree homogeneity of the cultured cell population and an evident reactivity of these cells to dBcAMP and FSH was achieved. Furthermore, an observation of giant cells, visible in monolayer among the typical Sertoli cells, is discussed in this paper. Contrary to small number of myoid cells, being survived in the culture, these giant cells did not show positive reaction to alkaline phosphatase.     

DIFFERENTIATION OF A PRIMARY CHEMICALLY INDUCED RAT NEPHROBLASTOMA IN ORGAN CULTURE

The differentiation in organ culture of a rat nephroblastoma is compared with differentiation of normal rat metanephric tissue under the same conditions. The nephroblastoma arose in a 19 week old female Fischer F344 rat given a single intraperitoneal injection of 4.0 μmole methyl(methoxymethy1)nitrosamine (DMN-OMe)/g body weight at one day of age. The tumor consisted almost entirely of spindle cells although a few well-differentiated tubules were scattered throughout the tumor mass. No primitive tubules were seen, but focal aggregates of tumor cells suggestive of nascent epithelial differentiation were frequent. Fragments of the nephroblastoma were cultured on gelfoam sponge in Williams Medium E supplemented with hydrocortisone, insulin, and fetal bovine serum. Within one day extensive tubulogenesis was observed. High mitotic activity resulted in a steady increase in the size of cultured explants over a period of 6 days. By day six, differentiating tubules filled the explant tissue. Cultured fragments were nearly indistinguishable histologically from normal F344 rat fetal kidney explanted to organ culture on day 15 of gestation and grown in vitro for the same period.     

Effects of an antiandrogen,cyproterone acetate,on the kidney of the three-spined stickleback (Gasterosteus aculeatus L.)

The fine structure of the kidney is studied in the sexually mature male three-spined stickleback after administration of an antiandrogen, cyproterone acetate. Under these conditions, the dedifferentiation of renal tubules is characterized by the same involutive processes as those induced by castration, with the difference that cyproterone acetate only begins to act after 14 days whereas after castration the first signs of involution are visible after 7 days. The ultrastructural modifications affect the nucleoli, the rough endoplasmic reticulum and the Golgi apparatus. They reflect an inhibition of the secretory process. The results obtained demonstrate that administration of cyproterone acetate to male sticklebacks has an inhibitory effect on renal target cells, apparently indistinguishable from the changes induced by lack of male sex hormone, and that this drug may be a valid substitute for castration in fish.     

Comparison of actin-filament bundles in myoid cells and Sertoli cells of the rat,golden hamster and mouse

Testes of adult rats, golden hamsters and mice were fixed with paraformaldehyde. Seminiferous tubules were then isolated by collagenase dissociation, stained with fluorescent phallotoxin, and viewed in a confocal laser microscope to observe actin filaments. Bundles of actin filaments in the myoid cells, especially in the rat, were arranged at right angles to each other in relation to the longitudinal axis of the tubule. In the hamster, circumferentially directed bundles were more frequent than longitudinally directed bundles. The actin bundles in the mouse were thinner than those in the rat and hamster, and their lattice network was less prominent. Nuclei of the myoid cells were elliptical and their short diameters were parallel to the long axis of the seminiferous tubules in the animals examined. Areas of myoid cells and of basal junctional portions of Sertoli cells were measured and compared in all animals studied. There were significant differences in the areas among the three species. The golden hamster showed the largest value for myoid-cell area, and the mean value for Sertoli-cell area was highest in the mouse.     

Cytochemical and biochemical characterization of testicular peritubular myoid cells

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.     

On the character of the monolayer outgrowth and the fate of the peritubular myoid cells in cultured mouse testis

Postnatal differentiation of the peritubular myoid cells in mouse testis is hormone dependent. In order to analyse the differentiation of the peritubular tissue, an attempt was made to develop an experimental model system utilizing an in vitro method. Fragments obtained from adult, 7- or 10-day-old mice, were cultured in McCoy's modified 5a medium for 9–19 days. The fragments and monolayers that grew from them were examined with the electron microscope at the end of the culture period. Monolayers originating from either mature or immature testicular expiants were comparable in appearance. They were composed of spindle-shaped cells that contained abundant profiles of granular endoplasmic reticulum and free ribosomes, as well as arrays of 40–60 Å thick filaments and associated dense bodies. In these respects they resembled smooth muscle cells in culture, in developmental, and in pathological conditions. Examination of the peritubular tissue in the testicular explants indicated that the monolayer of myoid cells originated from the fibroblasts rather than the peritubular myoid cells. Peritubular cells in explants from mature rats retained their myoid features at the end of the culture period but myoid cell differentiation failed to progress in expiants obtained from immature animals. Additional work is necessary in order to establish the suitability of these preliminary culture attempts to support normal development before conclusions may be drawn concerning the role of hormones in myoid cell differentiation. The role of microfilaments as a contractile organelle of cells is discussed.     

Modulation of alkaline phosphatase activity in alveolar type II like cells

Alveolar type II like cells (ALT II) represent a small subpopulation of alveolar type II cells, which is able to proliferate, can be passaged and possess many characteristics of differentiated adult type II cells. A correlation was found between the growth and development of ALT II cells in culture and their alkaline phosphatase activity. Unlike alveolar type II cells, which lose the activity in culture, ALT II cells regain the activity and maintain it for a long culture period. Quantitative histochemical analysis of the stained cells indicate that 80% of the cells at days 15-20 in culture are alkaline phosphatase positive. Inhibition studies indicate that alkaline phosphatase from ALT II cells and freshly isolated type II cells were similar. The inhibition of ALT II alkaline phosphatase by L-levamisole and its heat stability are similar to that of the bone enzyme and differ from the intestinal enzyme. Alkaline phosphatase expression is considered part of the differentiated phenotype of these cells. Therefore, the presence of this enzyme in ALT II cells adds support to the notion that these cells maintain many aspects of mature alveolar type II cells.     

Latent infection of epidermal Langerhans cells in HIV-positive individuals

Epidermal cell suspension otained fron 3 symptom-free HIV-positive individuals were cultured and marked with monoclonal antibodies for the HIV proteins p15, p24 and gp120 in the alkaline phosphatase anti-alkaline phosphatase staining technique. For 2 individuals, cells were positive after 3 days in culture, and for the third, after 4 days. Supernatant from one of the cultures infected allogeneic peripheral blood mononuclear cells. We conclude that epidermal Langerhans cells from symptom-free HIV-positive individuals are latent-infected and are able to produce and release HIV.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

Androgen nuclear exchange activity in rat testis.

Androgen nuclear exchange activity has been measured in the rat testis. Exchange activity was increased 2-10-fold by pretreatment of the tissue with testosterone and persisted for at least 21 days after hypophysectomy in mature animals. Competition for exchange activity using 3H-testosterone or 3H-dihydrotestosterone was significant for a 500-fold excess of testosterone, dihydrotestosterone, progesterone, and cyproterone acetate. The activity was found in tubules and in isolated germ cells from mature rat testes. These results suggest that the molecular mechanism of androgen action in the testis may be quite complex and may involve components both in the more mature germ cells as well.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat

Summary Staining of testicular and epididymal tissues after intravenous, intraperitoneal or subcutaneous administration of a number of dyes was investigated in rats at different stages of postnatal development. After light green injections heavy staining of both testis and epididymis was visible to the naked eye in neonatal animals up to the age of 10 days, while in rats over 15 days old no appreciable staining of the testis could be seen, although the caput epididymis was strongly coloured. From 3–8 hours after subcutaneous acriflavine administration, the nuclei in the blood vessel walls of the testis, as well as the nuclei in the rete testis, tubuli efferentes and caput epididymis, fluoresced in all age groups. The nuclei of the interstitial and tubular cells were stained intensely until the age of 5 days. Thereafter the intensity gradually diminished until the age of 20 days, when no nuclear fluorescence was visible in the seminiferous tubules and even the interstitial nuclei fluoresced weakly or not at all.The histochemical alkaline phosphatase activity of the testicular capillaries was studied by Gomori's method, using fresh and postfixed cryostat sections from postnatal rat testes. The testicular capillaries exhibited appreciable activity at the age of 10 days.On the basis of the present and previous observations on the permeability of the testicular capillaries, the existence of a blood-testis barrier in the puberal and adult rat testis is suggested.Development of the blood-testis barrier and the alkaline phosphatase activity of the testicular capillaries are suggested to reflect general vascular maturation at the beginning of puberty in the rat.Supported by grants from Yrjö Jahnsson's Foundation and P. O. Klingendahl Foundation.