A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.     

Role of sphingosine in the tumor necrosis factor alpha stimulatory effect on lactate dehydrogenase A expression and activity in porcine Sertoli cells

In this study, the intracellular signaling mechanisms through which TNFalpha increases LDH(A4) activity/expression in primary cultures of porcine testicular Sertoli cells were investigated. Studies were focused on sphingomyelin hydrolysis pathway. Treatment of [(14)C]serine-labeled cells with TNFalpha (15 ng/ml, 0.8 nM) resulted in a transient decrease (approximately 20%) in cellular [(14)C]sphingomyelin and in an increase (approximately 27%) in [(14)C]sphingosine that remained elevated for at least 75 min. In the same experiments, no significant changes were detected in ceramide levels. Exogenous sphingosine stimulated LDH(A4) activity and LDHA expression in a dose-dependent manner (ED(50) = 8 microM of sphingosine). Such an increase in LDHA messenger RNA levels and LDH(A4) activity was detected at 24 h and was maximal after 48 h of treatment. Kinetically, the increase in LDH(A4) activity was similar whether Sertoli cells were treated with sphingosine (12 microM) or with TNFalpha (20 ng/ml). Although sphingosine mimicked the action of TNFalpha on Sertoli cells LDH(A4) activity and expression, the maximal stimulatory effect represented about 30% of TNFalpha maximal activity. Sphingomyelinase, C2 ceramide, sphingosine 1-phosphate, N, N-dimethylsphingosine, and phosphorylcholine had no significant effect on LDHA expression/LDH(A4) activity. Exogenous C2 ceramide increased LDH(A4) activity only in cytokine-treated cells, suggesting its involvement as sphingosine precursor in TNFalpha-stimulated LDH(A4) activity via the sphingomyelin hydrolysis pathway. The LDH(A4) activity stimulated by TNFalpha was decreased by 36.2% by an inhibitor of sphingosine formation, NH4Cl (4 mM), supporting a role of sphingosine in the TNFalpha effect. Moreover, bisindolylmaleimide (100 nM), a protein kinase C (PKC) inhibitor decreased significantly by 28.7% the TNFalpha effect on LDH(A4) activity but had no effect on the stimulating action of sphingosine, suggesting that if PKC is involved in TNFalpha action, the sphingosine effect on LDH(A4) is unrelated to the PKC activity or inhibition. Together, the present data suggest that in primary Sertoli cell cultures, TNFalpha stimulating action on LDHA expression is partly exerted via sphingomyelin hydrolysis pathway, sphingosine being the active metabolite.     

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.     

Beta-nerve growth factor influences the expression of androgen-binding protein messenger ribonucleic acid in the rat testis.

The effect of infusion of nerve growth factor (NGF) into the rat testis on the expression of androgen-binding protein (ABP) mRNA was studied. A major 1.7-kb and a minor 3.7-kb ABP mRNA were present at all stages of the seminiferous epithelium with maximal levels at stages VIII-XI and the lowest levels at stages IV-VI. Infusion of 15 ng/h of NGF with a mini-osmotic pump for 14 days resulted in a 2-fold increase of ABP mRNA as revealed by Northern blots, whereas the mRNA level of another Sertoli cell protein, urokinase-type plasminogen activator, remained unchanged. Image analysis of autoradiograms obtained by in situ hybridization of sections from treated testes showed a similar increase in APB mRNA compared to noninfused or PBS-infused testes. However, at the cellular level the labeling intensity for ABP mRNA over Sertoli cells of different stages of the seminiferous epithelium was the same in NGF-infused and control testes. This suggests that the increase of ABP mRNA in NGF-infused testes was caused by prolongation of stages VII-VIII with maximal ABP mRNA expression; the suggestion is supported by an increase of 30 percent in frequency of these stages in histological sections from NGF-infused testes.     

Epidermal growth factor actions on arachidonic acid metabolism in human amnion cells

Epidermal growth factor (EGF) from human and murine sources increased prostaglandin E2 production by human amnion cells. An anti-EGF receptor monoclonal antibody abolished this stimulatory action of EGF and reduced the basal rate of amnion prostaglandin E2 biosynthesis. The rates of formation of arachidonate lipoxygenase products were also enhanced by EGF.     

Calcium/phospholipid-dependent protein kinases in rat Sertoli cells: regulation of androgen receptor messenger ribonucleic acid

The possibility that Sertoli cell responses to testosterone are modulated by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC) was examined in rat Sertoli cells in culture. Both soluble and particulate cell fractions showed low constitutive phosphotransferase activity. Incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) was associated with a transient induction in both cell fractions of calcium/phosphatidylserine-dependent PKC activity, which was elevated from 15 min to 1 h. Consistent with this, mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (alpha, beta, and gamma) were detected. The expression levels of mRNAs for PKCalpha and PKCbeta were also up-regulated (2.5- to 3-fold) by TPA (10(-7) M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10(-7) M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation ( approximately 70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at approximately 10(-8) M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.     

Effects of spinal cord injury on spermatogenesis and the expression of messenger ribonucleic acid for Sertoli cell proteins in rat Sertoli cell-enriched testes

The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes.     

Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat granulosa cells

We have investigated the effects of bFGF on both the FSH-induced LH receptor expression and cAMP production in cultured rat granulosa cells. Concentrations of pure FGF, from 10(-12) M to 10(-10) M, progressively inhibit the stimulatory actions of FSH with an ED50 of approximately 4 x 10(-12) M for both parameters. Higher FGF concentrations, from 4 x 10(-10) M to 10(-8) M, lead to a gradual reduction of the growth factor inhibitory effect. The effects of FGF are more prominent on the modulation of LH receptors than on the FSH-induced cAMP production. Moreover, FGF impairs the LH receptor formation induced by cholera toxin or 8-Bromo-cAMP, indicating that the growth factor also acts at a step distal to cAMP formation. The inhibitory effect of FGF on LH receptor expression increases during the entire course of granulosa cell differentiation, from 24 to 96 h, and is not due to variations in cell number or viability, but rather to a change in the content of LH receptors with no significant modification of binding affinity (KD congruent to 0.8 x 10(-10) M). These results suggest that bFGF may acutely regulate the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the ovarian follicular maturation.     

Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF-BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF-treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.     

Epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium in cultured porcine thyroid cells

The initial signal for thyroid cell proliferation is unknown. This is the first report to show that epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium ([Ca2+]i) in the thyroid gland. In cultured porcine thyroid cells, 10 nM EGF produces a breakdown of phosphatidylinositol and stimulates inositol phosphate production. Ten nM EGF increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the EGF-induced [Ca2+]i response occurs immediately, reaches a maximum within several seconds, and then slowly declines. EGF stimulates production of inositol phosphates, which seem to increase [Ca2+]i. Inositol phosphate production and an increase in [Ca2+]i after EGF-stimulation may function as an initial signal for thyroid cell proliferation.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor enhancement of insulin-like growth factor-I-induced down-regulation of insulin-like growth factor I receptor in cultured placental trophoblastic cells.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) synergistically stimulate placental lactogen (hPL) secretion by placental cells. To understand the mechanism of actions we have investigated a possible heterologous regulatory effect of EGF and IGF-I on each other's receptors. Pretreatment of the cells with IGF-I had no effect on [125I]-EGF binding or the down-regulation of EGF receptor. Pretreatment of the cells with EGF, concomitantly with IGF-I, had no effect on [125I]-IGF-I binding but it augmented the IGF-I down-regulation of IGF-I receptor. The time required to initiate the IGF-I-induced down-regulation of IGF-I receptor was reduced by 4 h in the presence of EGF. IGF-I-down-regulated decreased (P less than 0.05) receptor numbers were further decreased (p less than 0.05) in the presence of EGF. These results suggested that the synergistic effect of EGF and IGF-I seen in hPL secretion by placental cells is not due to direct heterologous hormone-receptor interactive effects. However, the effects seen may be due to a differentiating effect of EGF sensitizing the cells for responsiveness to IGF-I.     

Gonadotropin-releasing hormone stimulates annexin 5 messenger ribonucleic acid expression in the anterior pituitary cells

We previously reported that annexin 5 is found specifically in gonadotropes and that the expression is dramatically enhanced after ovariectomy. In the present study, the expression of annexin 5 was examined in the primary culture of rat anterior pituitary cells using semiquantitative RT-PCR to determine if it is under the direct control of gonadotropin-releasing hormone (GnRH). Continuous administration of GnRH analog for 1 h enhanced the expression of both FSH beta subunit and annexin 5 mRNA. The expression of annexin 5 mRNA was also augmented by phorbol 12-myristate 13-acetate but not by forskolin. Administration of recombinant rat annexin 5 to the culture increased LH beta mRNA expression. These data clearly demonstrate that the expression of annexin 5 mRNA is directly controlled by GnRH and suggest that annexin 5 is involved in mediating GnRH action in the pituitary gland.     

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.     

Increase in epidermal growth factor receptor and its messenger ribonucleic acid levels with differentiation of human trophoblast cells in culture

Epidermal growth factor receptor (EGFR) expression was studied during the differentiation of human trophoblast cells in culture. In vitro, intravillous mononuclear cytotrophoblasts aggregate and fuse within 24 h to form a syncytium. This morphological differentiation was associated with a significant twofold increase in specific ¹²⁵I-EGF binding capacity (P < 0.01). Scatchard analyses showed an apparent rise in the number of high-affinity binding sites (0.33 ± 0.04 and 0.63 ± 0.07 pmol/mg protein at 24 and 48 h, respectively), with no change in their affinity (1.34 and 1.42 × 10^?10 mol/L). Affinity labeling of ¹²⁵I-EGF in cultured trophoblast cells followed by SDS-PAGE and autoradiography revealed a band of 175 KDa corresponding to EGFR, the intensity of which increased with the time in culture. EGF-dependent phosphorylation of membrane proteins from cultured trophoblast cells revealed major phosphorylated proteins of 170 KDa (EGFR) and 35 KDa, which were both increased at 48 h, indicating a rise in EGFR-kinase activity during syncytium formation. Northern blot analysis of EGFR-mRNA, followed by hybridization with a ³²P-cDNA probe for EGFR, revealed an increase in EGFR gene expression in syncytiotrophoblasts, as compared to cytotrophoblasts. Thus, the increase in bioactive EGFR observed during the differentiation of trophoblast cells was due to an increase in their synthesis. Cultured trophoblast cells are therefore a good model of spontaneous up-regulation of EGFR expression with cell differentiation. © 1993 Wiley-Liss, Inc.