Purification of “colony-forming” cells from immature rat testis

Follicle stimulating hormone (FSH) enhances colony formation (as a result of reaggregation) by dissociated 10-day-old rat testis cells in primary culture. The purpose of this study was to examine various cytological characteristics of the FSH-responsive cells and develop techniques for their purification. The ability of testis cells to form colonies in response to FSH (5 μg/ml) was tested at various ages and was found to be maximal at 15 days of age. No colony formation occurred at ages greater than 20 days. Using colony formation as an assay for the FSH-responsive cells, techniques were developed for their purification. Colony cells were purified on a continuous bovine serum albumin (BSA) step gradient (1 ml/step). In fractions purified in this manner and subsequently cultured 24 h with FSH (5 μg/ml) 99.4% of attached cells were colony cells. Light and electron microscopy indicated that the colony cells (1) were one cell type; (2) were not germinal cells; (3) ultrastructurally resembled in situ Sertoli cell of the immature rat testis; and (4) contained a nucleolus with satellite karyosomes, structures which are characteristic of rat Sertoli cells. The mitotic index of the purified cells was 0.014% following 24 h in 10⁻⁵ M colchicine. Based on these data, it was concluded that the FSH responsive cells in culture are Sertoli cells.     

The effects of tunicamycin and 20-hydroxyecdysone on the cell surface properties of two insect cell lines

The radiolabeled lectins, concanavalin A* and wheat germ agglutinin, were used to study surface properties of two insect cell lines. We also looked at the effects of tunicamycin and 20-hydroxyecdysone on the binding of these lectins to one of the cell lines. Both UMBGE-2 and CH-MRRL cells bound both lectins, specifically. The CH-MRRL cells showed an overall higher binding for the lectins than the UMBGE-2 cells. This difference may account for some of the striking morphological difference seen between these cells. Tunicamycin and 20-hydroxyecdysone decreased the binding of both [¹²⁵I]-Con A and [¹²⁵I]-WGA to CH-MRRL cells. These results suggest that cell surface glycoproteins play a role in the modification of cellular morphology and in other hormone-mediated physiological functions.     

Effects of 20-hydroxyecdysone and tunicamycin on glycoprotein synthesis in an insect cell line

Treatment of CH-MRRL cells with either 20-hydroxyecdysone or tunicamycin resulted in a decrease in the incorporation of labeled sugars into glycoproteins. This change appears to be largely quantitative, as few qualitative changes in protein bands were apparent as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tunicamycin caused a greater change in the amount of labeled sugar incorporated into specific glycoproteins than did 20-hydroxyecdysone. This was more apparent in [¹⁴C]-mannose-labeled than in [¹⁴C]-N-acetylglucosamine-labeled glycoproteins. Both compounds caused changes in cell surface glycoproteins. These changes are discussed in relation to previous work on binding of lectins to the cell surface and on the mode of action of tunicamycin.     

Regulation of Sertoli cell cyclic adenosine 3':5' monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyrl cyclic AMP

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl cAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 ± 16 pmoles/min/mg protein; FSH: 291 ± 25 pmoles/min/mg protein; dibutyryl cAMP: 630 ± 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the induction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5–1.0 μg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterase activity via changes of cAMP levels in Sertoli cell in culture.     

A novel hypothalamic peptide, pituitary adenylate cyclase activating peptide, modulates Sertoli cell function in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP), a novel hypothalamic peptide that has been shown to exist in several tissues including the testis, was examined for its effects on cultured rat Sertoli cells. PACAP stimulates cAMP accumulation in Sertoli cells cultured from 15-day-old rats in the presence or absence of methylisobutylxanthine, a phosphodiesterase inhibitor, and in the presence of pertussis toxin, a blocker of the adenylate cyclase inhibitory pathway. Maximal stimulation, which is 20-40% of that attainable with FSH, occurs at PACAP concentrations of 10 nM: the ED50 is approximately 100 pM. The ability of PACAP to stimulate Sertoli cell cAMP declines with increasing age of donor animals (15-60 days of age) in a fashion similar to the FSH effect. PACAP stimulation of Sertoli cell cAMP accumulation is additive with submaximal, but not maximal, concentrations of FSH or forskolin. PACAP also stimulates the secretion of lactate, estradiol, and inhibin in a concentration-dependent manner. The stimulation of Sertoli cell cAMP accumulation by PACAP is not altered by a vasoactive intestinal peptide antagonist, and vasoactive intestinal peptide alone does not stimulate cAMP accumulation, indicating that PACAP is not acting via vasoactive intestinal peptide receptors. Further experiments are needed to determine whether PACAP is synthesized within the testis and if so, in which cell types; however, the present data clearly demonstrate that PACAP can modulate Sertoli cell function in vitro.     

The Sertoli cell in vivo and in vitro

The Sertoli cell extends from the basement membrane of the seminiferous tubule towards its lumen; it sends cytoplasmic processes which envelop different generations of germ cells. The use of Sertoli cell culture began to develop in 1975. To reduce germ cell contamination immature animals are generally used as Sertoli cell donors. Sertoli cell mitosis essentially occurs in sexually immature testes in mammals; mitosis of these cells is observed in vitro during a limited period of time. Sertoli cells in vivo perform an impressive range of functions: structural support of the seminiferous epithelium, displacement of germ cells and release of sperm; formation of the Sertoli cell blood-testis barrier; secretion of factors and nutrition of germ cells; phagocytosis of degenerating germ cells and of germ cell materials. Some of the Sertoli cell functions can be studied in vitro. The recent development of Sertoli cell culture on permeable supports (with or without extracellular matrix) has resulted in progress in understanding the vectorial secretion of several Sertoli cell markers. In addition to FSH and testosterone, several other humoral factors are known to influence Sertoli cell function. Furthermore, myoid cells bordering the tubules as well as germ cells are capable of regulating Sertoli cell activity. Sertoli cells are the most widely used testicular cells for in vitro toxicology. The testis is highly vulnerable to xenobiotics and radiations, yet the number of studies undertaken in this field is insufficient and should be drastically increased.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin

A cultured cell line of the mosquito, Aedes aegypti, is sensitive to tunicamycin as expected from the ability of crude membrane preparations to catalyse the formation of N-acetylglucosamine-linked dolichyl pyrophosphate. Formation of dolichylphosphomannose was also detected and this reaction was totally insensitive to tunicamycin. Incorporation of radioactive mannose into total acid-precipitable glycoproteins was inhibited greater than 90% in whole cells by tunicamycin, while the incorporation of leucine and glucosamine was less affected. Separation of the radioactive hexosamines from acid hydrolysates of cells incubated with [¹⁴C]glucosamine and tunicamycin showed predominant labelling of galactosamine, whereas in control cells not treated with the drug both glucosamine and galactosamine were labelled equally. Evidently, mosquito cells synthesise N-glycosidically linked carbohydrate chains assembled through tunicamycin-sensitive steps involving dolichyl pyrophospho-oligosaccharides, and O-glycosidically linked chains rich in N-acetylgalactosamine, the assembly of which is unaffected by tunicamycin. These results support structural evidence (Butters, T.D. and Hughes, R.C. (1981) Biochim. Biophys. Acta 640, 655–671) for the presence of high mannose N-glycans and N-acetylgalactosamine-richO-glycans in mosquito cell glycoproteins. The absence of complex N-glycans was confirmed by the demonstration of negligible activities of N-acetylglucosaminyl-, galactosyl- and sialyltransferases responsible for assembly of the terminal sequences of N-glycans of mature mammalian glycoproteins.     

Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat-germ agglutinin (WGA; N-acetyl-D-glucosamine-specific) at 50 μg/ml prevents the cell division of four-cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α-D-glucose and α-D-mannose-specific) treatment, 20 μg/ml, of four-cell or early eight-cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four-cell or early eight-cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α-L-fucose-specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid-specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5?IU/animal; T3 group-100?μg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

Inhibition of spontaneous aggregation by wheat germ agglutinin in suspensions of BALB/c-3T3 and BHK21 tissue culture fibroblasts

A quantitative method of measuring cytoaggregation based on the Coulter electronic cell counter has been applied to the agglutination of BALB/c-3T3 and BHK21 tissue culture fibroblasts by wheat germ agglutinin. When agglutinin is added to transformed or trypsinized cell suspensions high aggregation rates are seen, and the results obtained are in close agreement with the well-known sensitivity of transformed cells to agglutination by lectins.In the absence of agglutinin, a small but reproducible amount of spontaneous aggregation can also be detected. It is related in some way to growth, since it is absent in suspension prepared from confluent (density-inhibited) cultures and is induced by either transfer to low density, additional serum, or transformation by viruses. Under conditions favouring growth, both BALB/c-3T3 and BHK21 cells show aggregation indices close to 25, corresponding to 10% of the maximum aggregation rate seen.This spontaneous aggregation is susceptible to inhibition by agglutinin. Inhibition occurs at low concentration (about 10 μg/ml) and aggregation rates thus pass through a minimum as the concentration of agglutinin is increased. Among the four different cell lines studied, sensitivity to inhibition is inversely related to agglutination. Thus 3T3 cells, which are barely agglutinated by 1 mg/ml of agglutinin, show 90% inhibition; polyoma virus-transformed BHK cells, which are agglutinated by 10 μg of agglutinin, show no inhibition at all.It is suggested that the agglutination of transformed cells is a consequence of their failure to respond to an inhibitory effect exerted by lectins upon an intrinsic adhesive property of the cell surface.     

Kinetics of inhibin secretion in static and superfused Sertoli cell cultures in response to follicle-stimulating hormone

It is generally accepted that inhibin secretion in the testis is regulated by FSH; however, the kinetics of inhibin secretion have not been well defined in vivo and in vitro. We investigated the kinetics of inhibin secretion in response to FSH stimulation in static and superfused Sertoli cell cultures. Sertoli cells from 18-day-old rats were cultured in chemically defined medium for 3 days and were then stimulated for different time periods with FSH (0.1 microgram/ml). In static cultures, media were changed every 2, 4, or 8 h, and the superfusion was carried out at a steady rate of 3 ml/h. Inhibin in the culture media was measured by RIA, using antiserum against synthetic replicate [30Tyr]inhibin alpha-chain-(1-30) and, in some experiments, also by bioassay. The dynamics of inhibin secretion were similar in static and superfused Sertoli cell cultures. A significant increase (p less than 0.01) of inhibin secretion was noted after 5-6 h of FSH exposure. After 8-12 h of continuous FSH presence, the secretion of inhibin reached a maximal level, 5-10-fold higher than basal secretion (no FSH). In the continuous presence of FSH, inhibin secretion remained stable at the high level for up to 54 h. FSH removal caused a delayed (8-h) decrease (p less than 0.01) of inhibin secretion, with return to control basal values after approximately 30 h. When FSH was removed 4 h after its addition, inhibin secretion again increased 5-10-fold between 4 and 12 h, then returned to basal values within 30 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

The p38 MAPK pathway mediates interleukin-1-induced Sertoli cell proliferation

We have reported earlier that interleukin-1 (IL-1) is a potent growth factor for immature Sertoli cells (somatic cells in the testis required for testicular development and later spermatogenesis) and that this effect is synergistic with the mitogenic effect of follicle-stimulating hormone (FSH). The aim of the present study was to determine whether MAPK pathways are involved in mediating the mitogenic effect of IL-1 on Sertoli cells. Western blotting revealed that IL-1alpha activated p38 MAPK and JNK/SAPK, but not ERK, in Sertoli cells from 8- or 9-day-old rat. The inhibitor of p38 MAPK SB203580 attenuated the IL-1alpha-induced proliferation of Sertoli cells, as assessed by (3)H-thymidine incorporation and supravital staining as well as by direct cell counting. We conclude that the p38 MAPK pathway mediates the proliferative effect of IL-1alpha on immature Sertoli cells in vitro. Since the mitogenic effect of FSH is mediated via ERK, the synergistic action of IL-1alpha and FSH may be explained by their different intracellular signalling pathways. Induction of IL-1 by inflammation, infection or other tissue injuries may result in testicular damage by interfering with normal Sertoli cell development and thus future spermatogenesis.     

Studies on the mechanism of follicle-stimulating hormone-induced desensitization of Sertoli cell adenylyl cyclase in vitro

When Sertoli cells were cultured in the presence of follicle-stimulating hormone (FSH), a time-and concentration-dependent desensitization of FSH-responsive adenylyl cyclase (AC) was observed. Maximal desensitization (80%) was attained after 6-9 h of incubation with FSH (10 micrograms/ml; NIH-FSH-S12). During 24 h of incubation the concentration of FSH causing a half-maximal desensitization was about 100 ng/ml. Removal of the hormone from the culture medium was associated with a gradual reappearance of the FSH response. Follicle-stimulating hormone-induced desensitization of Sertoli cell AC was specific for homologous hormone, since AC activation by isoproterenol was unaffected. Furthermore, AC activity of control and FSH-desensitized cells was equally activated by GTP and fluoride, showing that the interaction of the guanyl nucleotide regulatory (N) component with the catalytic subunit is not affected during FSH-induced desensitization. A loss in specific FSH binding was detected after 9 and 24 h of exposure to FSH, but not at shorter times of incubation. Desensitization of Sertoli cell AC to both FSH and isoproterenol stimulation could also be achieved by dibutyryl cyclic AMP (dbcAMP); however, a 30-40% desensitization required a high nucleotide concentration (1 mM) and a long incubation time (24 h). These results show that desensitization of Sertoli cell AC by FSH is associated with normal function of the N component, and precedes any significant loss in specific FSH binding sites. Furthermore, exogenous addition of dbcAMP (1 mM) did not cause the same effects on Sertoli cell AC as did FSH.