- Email: [email protected]
THYROID-STIMULATING HORMONE AND GROWTH HORMONE RELEASE ALTERATIONS INDUCED BY MOSQUITO LARVAE PROTEINS ON PITUITARY CELLS
Cell Biology International 2001, Vol. 25, No. 9, 885–892 doi:10.1006/cbir.2001.0716, available online at http://www.idealibrary.com on
THYROID-STIMULATING HORMONE AND GROWTH HORMONE RELEASE ALTERATIONS INDUCED BY MOSQUITO LARVAE PROTEINS ON PITUITARY CELLS FEDERICO BOLOGNANI1,2, RODOLFO GUSTAVO GOYA1,2 and JORGE RAFAEL RONDEROS1 1
Ca´tedra de Histologia y Embriologı´a ‘B’, FCM—Ca´tedra de Histologia y Embriologia Animal, FCNyM—Universidad Nacional de La Plata, Calle 60 y 120 (1900), La Plata, Argentina 2 INIBIOLP, UNLP-CONICET, Calle 60 y 120 (1900), La Plata, Argentina Received 11 July 2000; accepted 5 January 2001
Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80 kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90C for 60 min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12 kD) on the concentration of both thyroid-stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time- and dose-dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90C for 60 min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator. 2001 Academic Press
K: insect; mosquito; growth factors; pituitary; thyroid stimulating hormone (TSH); growth hormone (GH).
INTRODUCTION Pituitary function is basically regulated by the action of messengers secreted at the hypothalamus level. It is also controlled by other types of molecules such as oestrogens and by growth factors acting in a paracrine, autocrine or even endocrine way. In fact, Finley et al. (1994) and Halper et al. (1992) have demonstrated the presence of several growth factors in the pituitary. Among others, members of the transforming growth factor To whom all correspondence should be addressed: Dr Jorge R. Ronderos, Ca´tedra de Histologı´a y Embriologı´a ‘B’, Fac. de Cs. Me´dicas-Universidad Nacional de La Plata, Calle 60 y 120 (1900) La Plata, Argentina. Fax: 054-0221-4258988; E-mail: jrondero@museo. fcnym.unlp.edu.ar 1065–6995/01/090885+08 $35.00/0
(TGF)- superfamily and its receptors have been described as regulators of growth and function of pituitary gland cell populations. Activins and inhibins play a very important role in the control of FSh and LH secretion (Woodruff and Mather, 1995). TGF-1 has also been referred to as a regulator of prolactotrope cell population, inhibiting proliferation and Prolactin (PRL) secretion in normal and oestrogenised rats (Albaladejo et al., 1992; Coya et al., 1999; De et al., 1996; Jin et al., 1997; Murata and Ying, 1991; Pastorcic et al., 1995; Sarkar et al., 1998). Moreover, the biological effects of the hormones can be also modulated by binding proteins, decreasing the effect by sequestering it, or increasing the biological action. 2001 Academic Press
886
These kind of factors and their receptors have been highly conserved throughout the evolutionary process. Thus, several genes codifying proteins with high homology with vertebrate growth factors and their receptors appear in nematodes, mollusks, and insects (Doctor et al., 1992; Massague, 1990; Muskavitch and Hoffmann, 1991; Padgett et al., 1987; Penton et al., 1997; Ren et al., 1996; Tin Xie et al., 1994). Previous experiments of our group have demonstrated that a crude extract from the larvae of the Culex pipiens L. mosquito modulates the proliferation of several epithelial cell populations of mice in vivo. The effect was either inhibitory at G1/S checkpoint or stimulatory at G2/M level in normal young mice, depending on the experimental design applied (Ronderos and Echave Llanos, 1990; Ronderos et al., 1994a; 1994b; Ronderos, 1996; Ronderos et al., 1996). The dialysed extract (12 kD cut-off pore membrane) also modulated proliferation of tongue keratinocytes and hepatocytes in adult hepatectomised mice (Ronderos et al., 1994a; 2000). After the assay of different soluble fractions of the extract isolated by molecular exclusion chromatography, one of them (with a molecular weight ranging from 12 to 80 kD, peaking at 25 kD) conserved the biological capabilities, inhibiting the mitotic rate of hepatocytes (Ronderos et al., 1999). Looking for an effect in cell populations other than epithelial ones, we assayed the extract on the proliferation of human mononuclear cells activated with the lectin, Concanavalin A. The response after 72 h culture was dosedependent and biphasic, inhibiting [3H]thymidine incorporation at higher doses and stimulating it at lower ones. Furthermore, when the extract was preheated at 90C for 60 min, the effect disappeared, suggesting that a protein was involved (Ronderos et al., 2000). The assay of soluble fractions of the extract isolated by molecular exclusion chromatography showed that the lower one (m.w. 12–80 kD) only inhibited DNA synthesis of human mononuclear cells (Ronderos et al., 2000). Based on the above information, we suggested the presence of some peptide resembling a TGF- superfamily activity in Culex pipiens mosquito larvae. As a first approach to the analysis of the insect protein effect on the anterior pituitary function, the action of the dialysed extract (12 kD cut-off pore membrane) of the mosquito larvae on TSH and GH release in dispersed cells of normal and oestrogenised pituitary rats was investigated.
Cell Biology International, Vol. 25, No. 9, 2001
MATERIAL AND METHODS Mosquito larvae extract Third and fourth instar larvae of Culex pipiens L. mosquito (Diptera: Culicidae) were obtained from an artificial colony. The larvae were separated from the colony, washed several times in fresh water to eliminate any breed-plate residues, then filtered and weighed for extract preparation. Pooled mosquito larvae were homogenised in a Potter-Dual homogeniser in an ice-bath and cold saline solution was added (2:10). The homogenate was centrifuged at 0C at 14,500 g for 20 min in a Sorvall RC5C centrifuge (crude extract) and then ultracentrifuged at 100,000 g for 60 min to remove microsomes. The supernatant was recovered and dialysed (12 kD cut-off pore membrane) in saline solution at 4C overnight. To assess thermostability, the extract was heated to 90C for 60 min, cooled in ice, and then centrifuged for 10 min. The protein content of the final preparation was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Animals Normal rats. Male Sprague-Dawley rats, aged 3–7 month-old (5 months average) were used. For each experiment an average of eight rats was used. The animals were kept under a 12:12 h light/dark regimen with water and food ad libitum. Oestrogenised rats. Female Sprague-Dawley rats, 7 months-old on average, were weekly injected i.m. with 0.2 ml 17-oestradiol valerate (0.2 mg/ml) in sunflower oil for 8 weeks. This procedure leads to the hyperplastic development of the somatotropes and prolactotrope cells (Ho et al., 1988; Oliveira et al., 1999). Anterior pituitary cell dispersion and incubation An average of eight anterior pituitaries were cut with a razor blade into 8–10 pieces each, and placed together in a Petri dish where they were washed twice with incubation medium (Earle’s Balanced Salt Solution (EBSS) containing 1 g/l glucose, 1 g/l NaCO3H, 0.5% bovine serum albumin, and 30 fg/ml ascorbic; pH 7.4). The pieces were transferred to a plastic tube containing 10 ml of medium with 32 mg collagenase type IV and 4 mg DNAse type I, to avoid cluster formation (Doyle et al., 1994). After 1 h incubation at 37C under constant shaking, the resulting cell
Cell Biology International, Vol. 25, No. 9, 2001
Statistical analysis Differences between treatments were analysed by one way analysis of variance. For single comparisons between means, Tukey Multiple Range Test was applied. Only differences of P<0.05 were considered significant. RESULTS TSH and GH Time-response in normal anterior pituitary cells With the aim of assaying for the first time the response of anterior pituitary dispersed cells to mosquito larvae dialysed extract and finding the optimal moment to analyse the effect, a time-course design was performed. After preincubating the dispersed pituitary cells as above described, the dialysed extract was added to the tubes at a final protein concentration of 250 g/ml. Incubation periods were 20, 40, 80, 120 and 160 min after treatments. The results showed an increment in the concentrations of TSH in the media as soon as 20 min after treatments. This increment was statistically significant and time-dependent, showing that higher concentrations of the hormones correlated with higher incubation times (Fig. 1). The range of variation in the increments between control and treated cells was from 142% (20 min) to 268% at 160 min. The dialysed extract was also active on GH concentration, in this case decreasing
160 * * 140
120
*
100 TSH (ng/ml)
suspension was gently dispersed by pippeting in order to eliminate cell clusters, and finally centrifuged at 500 g for 10 min. The supernatant was discarded and cells resuspended in fresh medium. Cell viability was evaluated by the Trypan blue exclusion test. The percentage of cell viability was always >90%. Cells were finally seeded at a concentration of 100,000 cells/ml. The cell dispersion was preincubated at 37C for 30 min in 750 l of medium and 250 l of medium containing the different doses of the dialysed extract was added (controls received medium only). Finally, the tubes containing the cells were centrifuged at 500 g for 10 min. The supernatants were recovered and frozen for radioimmunoassay (RIA). All the experiments were repeated at least twice. The quantity of released TSH and GH was assessed by RIA, with the materials obtained through the National Hormone and Pituitary Program (NHPP), NIDDK, NICHHD, USDA.
887
* 80
*
60
40
20
0
20
40
60 80 Time (minutes)
100
Fig. 1. Time-course curve for TSH secretion in pituitary cells from normal rats treated with 2.510 1 mg/ml of dialysed mosquito larvae extract. Increment of TSH concentration in the incubation medium was 153.71% (40 min) and 151.67% (80 min). Bars represent mean. *, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same. , basal; , treated.
it for all the times analysed (20–160 min of incubation; Fig. 2). The percentage of inhibition was extremely high (approx. 99% average). Taking into account these results we decide to choose 60 min incubation period for further experiments. Analysis of anterior pituitary cell viability after mosquito larvae dialysed extract assay Since a 60 min incubation period was chosen for further experiments, we checked cell viability after this interval of time. Four tubes receiving 250 g/ ml of mosquito larvae dialysed extract and four not receiving extract were recovered, and cell viability was evaluated. There were no differences in cell viability between controls and extract receiving cells, showing no lethal effect of the extract on the anterior pituitary cells.
888
Cell Biology International, Vol. 25, No. 9, 2001
180
10 000
160
*
140 1000
TSH (ng/ml)
100 *
100
80
60 * *
*
40
10
20
40
80 120 Time (minutes)
160
Fig. 2. Time-course curve for GH secretion in pituitary cells from normal rats treated with 2.510 1 mg/ml of dialysed mosquito larvae extract. Bars represent mean. *, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same. , basal;
, treated.
2.50E-01
5.00E-02
1.00E-02
2.00E-03
4.00E-04
8.00E-05
1.60E-05
3.20E-06
6.40E-07
* 0
1.28E-07
0
Heated
20
0
GH (log ng/ml)
120
Doses (mg/ml)
Fig. 3. Effect of dialysed extract on Tsh release and thermostability assay for the dialysed extract in dispersed pituitary cells obtained from normal rats. Doses of dialysed mosquito larvae extract ranged from 1.2810 7 to 2.510 1 mg/ml. Only a dose of 2.510 1 cause an increment of TSH secretion (126.3%, being this increment similar to the obtained in the time-response curve). Bars represent mean *, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same.
Effect of the dialysed extract in normal cells To evaluate further responses of the pituitary cells to the dialysed extract using the 60 min timecourse, a dose–response assay was performed. The dialysed extract was applied in different concentrations ranging from 2.510 1 to 1.310 7 mg/ml. As in the previous experiments, the treatments caused an increasing and a decreasing effect for TSH and GH concentration, respectively. The effect was statistically significant for a dose of 2.510 1 (TSH), and for doses ranging between 2.510 1 and 2.010 3 mg/ml (GH) (Figs 3 and 4). Radioimmunoassay interference In order to discard an eventual interference effect of the extract on the RIA used, and to ensure the reliability of the results obtained, a set of RIA
including mosquito larvae extract (250 g/ml) in the assay buffer was performed. Results showed that the extract does not produce a significant interference with TSH RIA, while it produces a minor overestimation for GH (data not shown). Furthermore, no TSH- or GH-like immunoreactivity was found in the extract by RIA (data not shown). Effect of temperature on biological activity of the extract With the aim of ruling out effects on the rat pituitary cells caused by steroid hormones normally present in insects, extracts assayed had previously been dialysed. To check the probable proteinaceous nature of the involved compounds, an experimental design including dialysed extract
Cell Biology International, Vol. 25, No. 9, 2001
889
10 000
10 000
1000
1000
*
*
* 100 *
100 *
10
10
previously heated at 90C for 60 min was performed. As an internal control, a set of four tubes was included with the highest concentration of the fresh extract, which previously caused a maximal increase or decrease of both hormones. Results showed that, while fresh extract significantly decreased GH concentration, the effect disappeared after heating (Fig. 4), as was also found for TSH secretion (Fig. 3). Effect of the dialysed extract on anterior pituitary cells from oestrogenised rats Prolactotrope and somatotrope cell populations are extremely sensitive to serial oestrogen treatments. This process normally induces the development of hyperplasia, resulting in an increment of PRL as well as GH secreting cells (Ho et al., 1988; Oliveira et al., 1999).
2.50E-01
5.00E-02
1.00E-02
2.00E-03
Doses (mg/ml)
Doses (mg/ml)
Fig. 4. Dose–response and thermolability assay for GH in pituitary cells from normal rats treated with doses of dialysed mosquito larvae extract ranging from 1.2810 7 to 2.510 1 mg/ml. Doses ranging from 2.510 1 to 2.010 3 mg/ml cause a decrease of GH concentration in the incubation medium. Bars represent mean*, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same.
4.00E-04
2.50E-01
5.00E-02
1.00E-02
2.00E-03
4.00E-04
8.00E-05
1.60E-05
3.20E-06
6.40E-07
1.28E-07
Heated
1
0
*
0
1
GH (log ng/ml)
GH (log ng/ml)
*
Fig. 5. Dose–response assay for GH in pituitary cells from oestrogenised rats treated with doses of dialysed mosquito larvae extract ranging from 4.010 4 to 2.510 1 mg/ml. Bars represent mean. *, P<0.05. Data are from a representative experiment of two performed. The effect observed was always the same.
Adult female Sprague-Dawley rats were treated with oestradiol-valerate as above described. When high PRL and GH concentrations were found in blood the rats were sacrificed and their pituitary glands were dissected and processed for incubation experiments. A dose–response assay was performed. The results obtained were similar to those with normal rats (i.e. a dose-dependent effect), showing an effect with doses ranging between 0.25 and 0.01 mg/ml (Fig. 5). DISCUSSION The present results show that mosquito larvae extract alters the concentration of GH and TSH in a rat anterior pituitary cells incubation system. These changes were not due to lethal toxic effects since viability assay showed no differences between control and treated cells after an incubation period of 60 min. The effect on both hormones proved to
890
be time- and dose-dependent. As treatments were performed with a 12 kD cut-off pore dialysed fraction, these results suggest that some macromolecule should be involved. The biological effect also disappeared after heating the extract at 90C, suggesting a proteinaceous nature of the biologically active molecule(s). Another interesting clue is that the effects elicited by mosquito preparations were opposite for both hormones analysed, showing increasing and decreasing concentrations of TSH and GH, respectively. This fact could be associated either with the presence of more than one compound acting independently or a simple one getting different effects on each of the two hormones analysed. The extract also proved to decrease the concentration of GH in cells isolated from oestrogenised rats. Several growth factors, e.g. EGF/TGT-, PDGF, IGF-I and others, are present in the pituitary gland (Finley et al., 1994; Halper et al., 1992). Furthermore, several members of the transforming growth factor- superfamily, such as TGF-1, activins and inhibins are also present in pituitary, acting as regulators of cell function. TGF-1 plays an important role inhibiting GH and PRL secretion, as well as prolactotrope cell proliferation (Albaladejo et al., 1992; Coya et al., 1999; De et al., 1996; Murata and Ying, 1991; Pastorcic et al., 1995; Sarkar et al., 1998). Activin, known to stimulate FSH secretion, also inhibits GH release, and was consequently proposed as another regulator of the somatotroph function (Murata and Ying, 1991; Bilezikjian et al., 1990; Kitaoka et al., 1988; Billestrup et al., 1990; Woodruff and Mather, 1995). In previous experiments, we had demonstrated that a soluble fraction of mosquito larvae extract having a 12–80 kD molecular weight (retentiontime peak at 25 kD) has a TGF--like effect on the mitotic rate of hepatocytes from hepatectomised mice (Ronderos et al., 1999). Some authors have reported the presence of immunologically positive cells for both GH and PRL in insects (Hansen et al., 1998; Schmid et al., 1989; Schmid et al., 1990; Swinnen et al., 1990; Veenstra et al., 1985), as well as the presence of genes codifying receptors structurally related to members of the TSH, LH/CG, FSH receptor family in mammals (Hauser et al., 1997). Several growth factors have been maintained throughout evolution. The presence of TGF-, activin, EGF and other vertebrate growth factors have been described in insects, nematodes, mollusks and other invertebrates by different methodologies (Franchini et al., 1996; Massague, 1990;
Cell Biology International, Vol. 25, No. 9, 2001
Muskavitch and Hoffmann, 1991; Doctor et al., 1992; Penton et al., 1997; Kutty et al., 1998; Padgett et al., 1987; Ren et al., 1996). Genes codifying for receptors of members of the TGF- superfamily have also been found in Drosophila (Tin Xie et al., 1994). Furthermore, TGF-1 and PDGF have proved to be related to immunocyte activity in molluscs (Kletsas et al., 1998; Ottaviani et al., 1997a; Ottaviani et al., 1997b). Experiments confirming the presence of members of the TGF- superfamily and/or the other vertebrates growth factors in Culex pipiens L. mosquito larvae are currently on the way. As shown above, the decrease of GH concentration in both normal and oestrogenised rat cells was extremely high. While the effect observed in TSH could be mediated by a stimulating effect on hormone release, the results observed for GH could not be explained only by this mechanism. Both, the high percentage of decrease for the hormone concentration and the short time elapsed between the treatment and the appearance of the effect, could be suggesting not only the presence of messengers but also the presence of some protein in mosquito larvae sequestering GH present in the incubation medium. Experiments to confirm this hypothesis are currently in progress. The chemical nature of these hormones and the cell populations secreting them are different. Furthermore, these effects seem to be the same for two other pituitary hormones which we are currently analysing, finding that the concentration of PRL (related to GH) is diminished, and the release of LH (related to TSH) is increased after mosquito protein treatments (Bolognani et al., 2001). Finally, our results suggest the presence of one or more compounds of polipeptidic nature modulating GH and TSH concentrations in the medium of pituitary cells from normal and oestrogenised rats. ACKNOWLEDGEMENTS The authors wish to thank Ms Gabriela Simonetto for editorial assistance, Ms Yolanda Sosa for technical assistance, and Dr Victoria Micieli from the Insect Pathology Laboratory (CEPAVE-UNLP) for mosquito larvae supply. REFERENCES A B, Z-L F, N B, J-P MO, A O, V M, A J, 1992. Possible involvement of Transforming Growth Factor- in the inhibition of rat
Cell Biology International, Vol. 25, No. 9, 2001
pituitary tumor growth by estradiol. J Esteroid Biochem Molec Biol 41: 125–134. B LM, C AZ, V W, 1990. Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells. Endocrinology 126: 2369–2376. B N, G-M C, P E, V W, 1990. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 4: 356–362. B F, G RG, R JR, 2001. Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats. Cell Biol Int 25: 138–146. C R, A CV, P F, G C, D C, 1999. Effect of TGF-1 on prolactin synthesis and secretion: An in vitro study. J Neuroendocrinol 11: 351–360. D A, M TE, S RC, B N, S DK, 1996. Pituitary lactotrope express transforming growth factor (TGF) type II receptor mRNA and protein and contains 125I-TGF1 binding sites. J Endocrinol 149: 19–27. D JS, J PD, R KE, V M, H FM, 1992. Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster. Dev Biol 151: 491– 505. D A, G JB, N DG, 1994. Cell & Tissue Culture: Laboratory Procedures. Ed. by ECACC. London, John Wiley and Sons. F EL, K JS, R JS, 1994. Human pituitary somatotropes express Transforming Growth Factor- and its receptor. J Endocrinol 141: 547–554. F A, K D, O E, 1996. Immunocytochemical evidence of PDGF- and TGF--like molecules in invertebrate and vertebrate immunocytes: an evolutionary approach. Histochem J 28: 599–603. H J, P PG, C BJ, R P, S BW, 1992. Presence of growth factors in human pituitary. Lab Inv 66: 639–645. H GN, H BL, S B, 1988. Diversity of prolactin systems in the insect Leucophaea maderae: use of antiserum polyclonality for immunocytochemical detection of neuropeptide heterogeneity. Cell Tissue Res 252: 557–563. H F, N HP, G CJP, 1997. Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the ThyroidStimulating Hormone, Follicle-Stimulating Hormone, Luteinizing Hormone/Choriogonadotropin receptor family from mammals. J Biol Chem 272: 1002–1010. H KY, T MO, K RJ, L SK, S YN, J ML, L DA, E WS, 1988. Effects of gonadal steroids on somatotroph function in the rat: analysis by the reverse hemolytic plaque assay. Endocrinology 123: 1405–1411. J L, Q X, K E, S N, S BW, K K, Y WF, L RV, 1997. Transforming growth Factor-, Transforming growth Factor- receptor I, and p27Kip1 expression in nontumorous and neoplastic human pituitaries. Am J Pathol 151: 509–519. K M, K I, O E, 1988. Activin-A: A modulator of multiple types of anterior pituitary cells. Biochem Biophys Res Commun 157: 48–54. K D, S D, F A, O E, 1998. PDGF and TGF- induce cell shape changes in invertebrates immunocytes via specific cell surface receptors. Eur J Cell Biol 75: 362–366.
891
K G, K RK, S W, D T, J C, W B, 1998. Identification of a new member of Transforming Growth Factor-Beta superfamily in Drosophila: the first invertebrate activin gene. Biochem Biophy Res Comm 246: 644–649. L OH, R NJ, F AL, R RJ, 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275. M J, 1990. The Transforming Growth Factor- family. Annu Rev Cell Biol 6: 597–641. M T, Y S, 1991. Transforming Growth Factor- and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system. Proc Soc Exp Biol Med 198: 599–605. M MA, H FM, 1991. Homologs of vertebrate growth factors in Drosophila melanogaster and other invertebrates. Curr Tops Dev Biol 24: 289–327. O MC, M HP, T M, BC L, 1999. Chronic effect of antidopaminergic drugs or estrogen on male Wistar rat lactotrophs and somatotrophs. Braz J Med Biol Res 32: 1561–1564. O E, F A, K D, B M, G S, 1997a. Involvement of PDGF and TGF- in cell migration and phagocytosis in invertebrate and human immunocytes. Anim Biol 6: 91–95. O E, S D, K D, 1997b. PDGF- and TGF-induced changes in cell shape of invertebrate immunocytes: effect of calcium entry blockers. Eur J Cell Biol 74: 336–341. P RW, S J RD, G WM, 1987. A transcript from Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature 325: 81–84. P M, D A, B N, V W, S DK, 1995. Reduction in the expression and action of transforming growth factor beta 1 on lactotropes during estrogeninduced tumorigenesis in the anterior pituitary. Cancer Res 55: 4892–4898. P A, S SB, H FM, 1997. Regulation of cell cycles synchronization by decapentaplegic during Drosophila eye development. Science 275: 203–206. R P, L C, J R, A PS, P D, R DL, 1996. Control of C. elegans larval development by neuronal expression of a TGF- homologue. Science 274: 1389–1391. R JR, E L JM, 1990. Inhibiting effect of mosquito larvae extract on the mitotic rate of hepatocytes of young growing and adult partially hepatectomized mice. Com Biol 9: 133–142. R JR, S AO, A CA, E L JM, 1994a. Inhibition of mouse parenchymal cells proliferation by mosquito larvae extract. Cell Mol Biol 40: 1183– 1188. R JR, O A, S A, E L JM, 1994b. Further action of mosquito larvae extract on the mitotic rate of mouse cell populations. BioCell 18: 51–57. R JR, 1996. Early effect of the mosquito larvae extract on the mouse cells proliferation. Cell Biol Int 20: 249–254. R JR, R FL, O AC, 1996. Mosquito larvae extract induces mitotic rate alterations of mice tongue keratinocytes along a circadian rhythm period. Cell Biol Int 20: 567–572. R JR, R OJ, S MA, B RR, 1999. Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation. Cell Biol Int 23: 219–226.
892
R JR, S MA, R OJ, F G, 2000. Partial characterisation of mosquito larvae extract inducing DNA synthesis alterations on human mononuclear cells. Cell Biol Int 24: 175–181. S D, P M, D A, E M, M H, G MB, 1998. Role of Transforming Growth Factor (TGF)- Type I and TGF- type II receptors in the TGF-I-regulated gene expression in pituitary prolactinsecreting lactotropes. Endocrinology 139: 3620–3628. S KP, M V, O B, P EF, 1989. Immunocytochemical localization of prolactin-like antigenic determinants in the neuroendocrine system of the honeybee (Apis mellifica). Histochemistry 91: 469–472. S KP, M V, H C, P EF, 1990. Ultrastructural localization of prolactin-like antigenic determinants in neurosecretory cells in the brain of the honeybee (Apis mellifica). Horm Metab Res 22: 413–417.
Cell Biology International, Vol. 25, No. 9, 2001
S K, B JV, V P, D L A, 1990. Immunocytochemical localization of human growth hormone- and prolactin-like antigenic determinants in the insects, Locusta migratoria and Sarcophaga bullata. Comp Biochem Physiol A 95: 373–378. T X, F AL, P RW, 1994. The Drosophila saxophone gene: A serine-Threonine kinase receptor of the TGF-beta superfamily. Science 263: 1756–1759. V JA, R-P HM, S H, P JM, 1985. Immunocytochemical localization of peptidergic neuron and neurosecretory cells in the neuroendocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides. Histochemistry 82: 9–18. W TK, M JP, 1995. Inhibin, activin and the female reproductive axis. Ann Rev Physiol 57: 219–224.
THYROID-STIMULATING HORMONE AND GROWTH HORMONE RELEASE ALTERATIONS INDUCED BY MOSQUITO LARVAE PROTEINS ON PITUITARY CELLS FEDERICO BOLOGNANI1,2, RODOLFO GUSTAVO GOYA1,2 and JORGE RAFAEL RONDEROS1 1
Ca´tedra de Histologia y Embriologı´a ‘B’, FCM—Ca´tedra de Histologia y Embriologia Animal, FCNyM—Universidad Nacional de La Plata, Calle 60 y 120 (1900), La Plata, Argentina 2 INIBIOLP, UNLP-CONICET, Calle 60 y 120 (1900), La Plata, Argentina Received 11 July 2000; accepted 5 January 2001
Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80 kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90C for 60 min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12 kD) on the concentration of both thyroid-stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time- and dose-dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90C for 60 min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator. 2001 Academic Press
K: insect; mosquito; growth factors; pituitary; thyroid stimulating hormone (TSH); growth hormone (GH).
INTRODUCTION Pituitary function is basically regulated by the action of messengers secreted at the hypothalamus level. It is also controlled by other types of molecules such as oestrogens and by growth factors acting in a paracrine, autocrine or even endocrine way. In fact, Finley et al. (1994) and Halper et al. (1992) have demonstrated the presence of several growth factors in the pituitary. Among others, members of the transforming growth factor To whom all correspondence should be addressed: Dr Jorge R. Ronderos, Ca´tedra de Histologı´a y Embriologı´a ‘B’, Fac. de Cs. Me´dicas-Universidad Nacional de La Plata, Calle 60 y 120 (1900) La Plata, Argentina. Fax: 054-0221-4258988; E-mail: jrondero@museo. fcnym.unlp.edu.ar 1065–6995/01/090885+08 $35.00/0
(TGF)- superfamily and its receptors have been described as regulators of growth and function of pituitary gland cell populations. Activins and inhibins play a very important role in the control of FSh and LH secretion (Woodruff and Mather, 1995). TGF-1 has also been referred to as a regulator of prolactotrope cell population, inhibiting proliferation and Prolactin (PRL) secretion in normal and oestrogenised rats (Albaladejo et al., 1992; Coya et al., 1999; De et al., 1996; Jin et al., 1997; Murata and Ying, 1991; Pastorcic et al., 1995; Sarkar et al., 1998). Moreover, the biological effects of the hormones can be also modulated by binding proteins, decreasing the effect by sequestering it, or increasing the biological action. 2001 Academic Press
886
These kind of factors and their receptors have been highly conserved throughout the evolutionary process. Thus, several genes codifying proteins with high homology with vertebrate growth factors and their receptors appear in nematodes, mollusks, and insects (Doctor et al., 1992; Massague, 1990; Muskavitch and Hoffmann, 1991; Padgett et al., 1987; Penton et al., 1997; Ren et al., 1996; Tin Xie et al., 1994). Previous experiments of our group have demonstrated that a crude extract from the larvae of the Culex pipiens L. mosquito modulates the proliferation of several epithelial cell populations of mice in vivo. The effect was either inhibitory at G1/S checkpoint or stimulatory at G2/M level in normal young mice, depending on the experimental design applied (Ronderos and Echave Llanos, 1990; Ronderos et al., 1994a; 1994b; Ronderos, 1996; Ronderos et al., 1996). The dialysed extract (12 kD cut-off pore membrane) also modulated proliferation of tongue keratinocytes and hepatocytes in adult hepatectomised mice (Ronderos et al., 1994a; 2000). After the assay of different soluble fractions of the extract isolated by molecular exclusion chromatography, one of them (with a molecular weight ranging from 12 to 80 kD, peaking at 25 kD) conserved the biological capabilities, inhibiting the mitotic rate of hepatocytes (Ronderos et al., 1999). Looking for an effect in cell populations other than epithelial ones, we assayed the extract on the proliferation of human mononuclear cells activated with the lectin, Concanavalin A. The response after 72 h culture was dosedependent and biphasic, inhibiting [3H]thymidine incorporation at higher doses and stimulating it at lower ones. Furthermore, when the extract was preheated at 90C for 60 min, the effect disappeared, suggesting that a protein was involved (Ronderos et al., 2000). The assay of soluble fractions of the extract isolated by molecular exclusion chromatography showed that the lower one (m.w. 12–80 kD) only inhibited DNA synthesis of human mononuclear cells (Ronderos et al., 2000). Based on the above information, we suggested the presence of some peptide resembling a TGF- superfamily activity in Culex pipiens mosquito larvae. As a first approach to the analysis of the insect protein effect on the anterior pituitary function, the action of the dialysed extract (12 kD cut-off pore membrane) of the mosquito larvae on TSH and GH release in dispersed cells of normal and oestrogenised pituitary rats was investigated.
Cell Biology International, Vol. 25, No. 9, 2001
MATERIAL AND METHODS Mosquito larvae extract Third and fourth instar larvae of Culex pipiens L. mosquito (Diptera: Culicidae) were obtained from an artificial colony. The larvae were separated from the colony, washed several times in fresh water to eliminate any breed-plate residues, then filtered and weighed for extract preparation. Pooled mosquito larvae were homogenised in a Potter-Dual homogeniser in an ice-bath and cold saline solution was added (2:10). The homogenate was centrifuged at 0C at 14,500 g for 20 min in a Sorvall RC5C centrifuge (crude extract) and then ultracentrifuged at 100,000 g for 60 min to remove microsomes. The supernatant was recovered and dialysed (12 kD cut-off pore membrane) in saline solution at 4C overnight. To assess thermostability, the extract was heated to 90C for 60 min, cooled in ice, and then centrifuged for 10 min. The protein content of the final preparation was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Animals Normal rats. Male Sprague-Dawley rats, aged 3–7 month-old (5 months average) were used. For each experiment an average of eight rats was used. The animals were kept under a 12:12 h light/dark regimen with water and food ad libitum. Oestrogenised rats. Female Sprague-Dawley rats, 7 months-old on average, were weekly injected i.m. with 0.2 ml 17-oestradiol valerate (0.2 mg/ml) in sunflower oil for 8 weeks. This procedure leads to the hyperplastic development of the somatotropes and prolactotrope cells (Ho et al., 1988; Oliveira et al., 1999). Anterior pituitary cell dispersion and incubation An average of eight anterior pituitaries were cut with a razor blade into 8–10 pieces each, and placed together in a Petri dish where they were washed twice with incubation medium (Earle’s Balanced Salt Solution (EBSS) containing 1 g/l glucose, 1 g/l NaCO3H, 0.5% bovine serum albumin, and 30 fg/ml ascorbic; pH 7.4). The pieces were transferred to a plastic tube containing 10 ml of medium with 32 mg collagenase type IV and 4 mg DNAse type I, to avoid cluster formation (Doyle et al., 1994). After 1 h incubation at 37C under constant shaking, the resulting cell
Cell Biology International, Vol. 25, No. 9, 2001
Statistical analysis Differences between treatments were analysed by one way analysis of variance. For single comparisons between means, Tukey Multiple Range Test was applied. Only differences of P<0.05 were considered significant. RESULTS TSH and GH Time-response in normal anterior pituitary cells With the aim of assaying for the first time the response of anterior pituitary dispersed cells to mosquito larvae dialysed extract and finding the optimal moment to analyse the effect, a time-course design was performed. After preincubating the dispersed pituitary cells as above described, the dialysed extract was added to the tubes at a final protein concentration of 250 g/ml. Incubation periods were 20, 40, 80, 120 and 160 min after treatments. The results showed an increment in the concentrations of TSH in the media as soon as 20 min after treatments. This increment was statistically significant and time-dependent, showing that higher concentrations of the hormones correlated with higher incubation times (Fig. 1). The range of variation in the increments between control and treated cells was from 142% (20 min) to 268% at 160 min. The dialysed extract was also active on GH concentration, in this case decreasing
160 * * 140
120
*
100 TSH (ng/ml)
suspension was gently dispersed by pippeting in order to eliminate cell clusters, and finally centrifuged at 500 g for 10 min. The supernatant was discarded and cells resuspended in fresh medium. Cell viability was evaluated by the Trypan blue exclusion test. The percentage of cell viability was always >90%. Cells were finally seeded at a concentration of 100,000 cells/ml. The cell dispersion was preincubated at 37C for 30 min in 750 l of medium and 250 l of medium containing the different doses of the dialysed extract was added (controls received medium only). Finally, the tubes containing the cells were centrifuged at 500 g for 10 min. The supernatants were recovered and frozen for radioimmunoassay (RIA). All the experiments were repeated at least twice. The quantity of released TSH and GH was assessed by RIA, with the materials obtained through the National Hormone and Pituitary Program (NHPP), NIDDK, NICHHD, USDA.
887
* 80
*
60
40
20
0
20
40
60 80 Time (minutes)
100
Fig. 1. Time-course curve for TSH secretion in pituitary cells from normal rats treated with 2.510 1 mg/ml of dialysed mosquito larvae extract. Increment of TSH concentration in the incubation medium was 153.71% (40 min) and 151.67% (80 min). Bars represent mean. *, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same. , basal; , treated.
it for all the times analysed (20–160 min of incubation; Fig. 2). The percentage of inhibition was extremely high (approx. 99% average). Taking into account these results we decide to choose 60 min incubation period for further experiments. Analysis of anterior pituitary cell viability after mosquito larvae dialysed extract assay Since a 60 min incubation period was chosen for further experiments, we checked cell viability after this interval of time. Four tubes receiving 250 g/ ml of mosquito larvae dialysed extract and four not receiving extract were recovered, and cell viability was evaluated. There were no differences in cell viability between controls and extract receiving cells, showing no lethal effect of the extract on the anterior pituitary cells.
888
Cell Biology International, Vol. 25, No. 9, 2001
180
10 000
160
*
140 1000
TSH (ng/ml)
100 *
100
80
60 * *
*
40
10
20
40
80 120 Time (minutes)
160
Fig. 2. Time-course curve for GH secretion in pituitary cells from normal rats treated with 2.510 1 mg/ml of dialysed mosquito larvae extract. Bars represent mean. *, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same. , basal;
, treated.
2.50E-01
5.00E-02
1.00E-02
2.00E-03
4.00E-04
8.00E-05
1.60E-05
3.20E-06
6.40E-07
* 0
1.28E-07
0
Heated
20
0
GH (log ng/ml)
120
Doses (mg/ml)
Fig. 3. Effect of dialysed extract on Tsh release and thermostability assay for the dialysed extract in dispersed pituitary cells obtained from normal rats. Doses of dialysed mosquito larvae extract ranged from 1.2810 7 to 2.510 1 mg/ml. Only a dose of 2.510 1 cause an increment of TSH secretion (126.3%, being this increment similar to the obtained in the time-response curve). Bars represent mean *, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same.
Effect of the dialysed extract in normal cells To evaluate further responses of the pituitary cells to the dialysed extract using the 60 min timecourse, a dose–response assay was performed. The dialysed extract was applied in different concentrations ranging from 2.510 1 to 1.310 7 mg/ml. As in the previous experiments, the treatments caused an increasing and a decreasing effect for TSH and GH concentration, respectively. The effect was statistically significant for a dose of 2.510 1 (TSH), and for doses ranging between 2.510 1 and 2.010 3 mg/ml (GH) (Figs 3 and 4). Radioimmunoassay interference In order to discard an eventual interference effect of the extract on the RIA used, and to ensure the reliability of the results obtained, a set of RIA
including mosquito larvae extract (250 g/ml) in the assay buffer was performed. Results showed that the extract does not produce a significant interference with TSH RIA, while it produces a minor overestimation for GH (data not shown). Furthermore, no TSH- or GH-like immunoreactivity was found in the extract by RIA (data not shown). Effect of temperature on biological activity of the extract With the aim of ruling out effects on the rat pituitary cells caused by steroid hormones normally present in insects, extracts assayed had previously been dialysed. To check the probable proteinaceous nature of the involved compounds, an experimental design including dialysed extract
Cell Biology International, Vol. 25, No. 9, 2001
889
10 000
10 000
1000
1000
*
*
* 100 *
100 *
10
10
previously heated at 90C for 60 min was performed. As an internal control, a set of four tubes was included with the highest concentration of the fresh extract, which previously caused a maximal increase or decrease of both hormones. Results showed that, while fresh extract significantly decreased GH concentration, the effect disappeared after heating (Fig. 4), as was also found for TSH secretion (Fig. 3). Effect of the dialysed extract on anterior pituitary cells from oestrogenised rats Prolactotrope and somatotrope cell populations are extremely sensitive to serial oestrogen treatments. This process normally induces the development of hyperplasia, resulting in an increment of PRL as well as GH secreting cells (Ho et al., 1988; Oliveira et al., 1999).
2.50E-01
5.00E-02
1.00E-02
2.00E-03
Doses (mg/ml)
Doses (mg/ml)
Fig. 4. Dose–response and thermolability assay for GH in pituitary cells from normal rats treated with doses of dialysed mosquito larvae extract ranging from 1.2810 7 to 2.510 1 mg/ml. Doses ranging from 2.510 1 to 2.010 3 mg/ml cause a decrease of GH concentration in the incubation medium. Bars represent mean*, P<0.05. Data are from a representative experiment of three performed. The effect observed was always the same.
4.00E-04
2.50E-01
5.00E-02
1.00E-02
2.00E-03
4.00E-04
8.00E-05
1.60E-05
3.20E-06
6.40E-07
1.28E-07
Heated
1
0
*
0
1
GH (log ng/ml)
GH (log ng/ml)
*
Fig. 5. Dose–response assay for GH in pituitary cells from oestrogenised rats treated with doses of dialysed mosquito larvae extract ranging from 4.010 4 to 2.510 1 mg/ml. Bars represent mean. *, P<0.05. Data are from a representative experiment of two performed. The effect observed was always the same.
Adult female Sprague-Dawley rats were treated with oestradiol-valerate as above described. When high PRL and GH concentrations were found in blood the rats were sacrificed and their pituitary glands were dissected and processed for incubation experiments. A dose–response assay was performed. The results obtained were similar to those with normal rats (i.e. a dose-dependent effect), showing an effect with doses ranging between 0.25 and 0.01 mg/ml (Fig. 5). DISCUSSION The present results show that mosquito larvae extract alters the concentration of GH and TSH in a rat anterior pituitary cells incubation system. These changes were not due to lethal toxic effects since viability assay showed no differences between control and treated cells after an incubation period of 60 min. The effect on both hormones proved to
890
be time- and dose-dependent. As treatments were performed with a 12 kD cut-off pore dialysed fraction, these results suggest that some macromolecule should be involved. The biological effect also disappeared after heating the extract at 90C, suggesting a proteinaceous nature of the biologically active molecule(s). Another interesting clue is that the effects elicited by mosquito preparations were opposite for both hormones analysed, showing increasing and decreasing concentrations of TSH and GH, respectively. This fact could be associated either with the presence of more than one compound acting independently or a simple one getting different effects on each of the two hormones analysed. The extract also proved to decrease the concentration of GH in cells isolated from oestrogenised rats. Several growth factors, e.g. EGF/TGT-, PDGF, IGF-I and others, are present in the pituitary gland (Finley et al., 1994; Halper et al., 1992). Furthermore, several members of the transforming growth factor- superfamily, such as TGF-1, activins and inhibins are also present in pituitary, acting as regulators of cell function. TGF-1 plays an important role inhibiting GH and PRL secretion, as well as prolactotrope cell proliferation (Albaladejo et al., 1992; Coya et al., 1999; De et al., 1996; Murata and Ying, 1991; Pastorcic et al., 1995; Sarkar et al., 1998). Activin, known to stimulate FSH secretion, also inhibits GH release, and was consequently proposed as another regulator of the somatotroph function (Murata and Ying, 1991; Bilezikjian et al., 1990; Kitaoka et al., 1988; Billestrup et al., 1990; Woodruff and Mather, 1995). In previous experiments, we had demonstrated that a soluble fraction of mosquito larvae extract having a 12–80 kD molecular weight (retentiontime peak at 25 kD) has a TGF--like effect on the mitotic rate of hepatocytes from hepatectomised mice (Ronderos et al., 1999). Some authors have reported the presence of immunologically positive cells for both GH and PRL in insects (Hansen et al., 1998; Schmid et al., 1989; Schmid et al., 1990; Swinnen et al., 1990; Veenstra et al., 1985), as well as the presence of genes codifying receptors structurally related to members of the TSH, LH/CG, FSH receptor family in mammals (Hauser et al., 1997). Several growth factors have been maintained throughout evolution. The presence of TGF-, activin, EGF and other vertebrate growth factors have been described in insects, nematodes, mollusks and other invertebrates by different methodologies (Franchini et al., 1996; Massague, 1990;
Cell Biology International, Vol. 25, No. 9, 2001
Muskavitch and Hoffmann, 1991; Doctor et al., 1992; Penton et al., 1997; Kutty et al., 1998; Padgett et al., 1987; Ren et al., 1996). Genes codifying for receptors of members of the TGF- superfamily have also been found in Drosophila (Tin Xie et al., 1994). Furthermore, TGF-1 and PDGF have proved to be related to immunocyte activity in molluscs (Kletsas et al., 1998; Ottaviani et al., 1997a; Ottaviani et al., 1997b). Experiments confirming the presence of members of the TGF- superfamily and/or the other vertebrates growth factors in Culex pipiens L. mosquito larvae are currently on the way. As shown above, the decrease of GH concentration in both normal and oestrogenised rat cells was extremely high. While the effect observed in TSH could be mediated by a stimulating effect on hormone release, the results observed for GH could not be explained only by this mechanism. Both, the high percentage of decrease for the hormone concentration and the short time elapsed between the treatment and the appearance of the effect, could be suggesting not only the presence of messengers but also the presence of some protein in mosquito larvae sequestering GH present in the incubation medium. Experiments to confirm this hypothesis are currently in progress. The chemical nature of these hormones and the cell populations secreting them are different. Furthermore, these effects seem to be the same for two other pituitary hormones which we are currently analysing, finding that the concentration of PRL (related to GH) is diminished, and the release of LH (related to TSH) is increased after mosquito protein treatments (Bolognani et al., 2001). Finally, our results suggest the presence of one or more compounds of polipeptidic nature modulating GH and TSH concentrations in the medium of pituitary cells from normal and oestrogenised rats. ACKNOWLEDGEMENTS The authors wish to thank Ms Gabriela Simonetto for editorial assistance, Ms Yolanda Sosa for technical assistance, and Dr Victoria Micieli from the Insect Pathology Laboratory (CEPAVE-UNLP) for mosquito larvae supply. REFERENCES A B, Z-L F, N B, J-P MO, A O, V M, A J, 1992. Possible involvement of Transforming Growth Factor- in the inhibition of rat
Cell Biology International, Vol. 25, No. 9, 2001
pituitary tumor growth by estradiol. J Esteroid Biochem Molec Biol 41: 125–134. B LM, C AZ, V W, 1990. Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells. Endocrinology 126: 2369–2376. B N, G-M C, P E, V W, 1990. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 4: 356–362. B F, G RG, R JR, 2001. Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats. Cell Biol Int 25: 138–146. C R, A CV, P F, G C, D C, 1999. Effect of TGF-1 on prolactin synthesis and secretion: An in vitro study. J Neuroendocrinol 11: 351–360. D A, M TE, S RC, B N, S DK, 1996. Pituitary lactotrope express transforming growth factor (TGF) type II receptor mRNA and protein and contains 125I-TGF1 binding sites. J Endocrinol 149: 19–27. D JS, J PD, R KE, V M, H FM, 1992. Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster. Dev Biol 151: 491– 505. D A, G JB, N DG, 1994. Cell & Tissue Culture: Laboratory Procedures. Ed. by ECACC. London, John Wiley and Sons. F EL, K JS, R JS, 1994. Human pituitary somatotropes express Transforming Growth Factor- and its receptor. J Endocrinol 141: 547–554. F A, K D, O E, 1996. Immunocytochemical evidence of PDGF- and TGF--like molecules in invertebrate and vertebrate immunocytes: an evolutionary approach. Histochem J 28: 599–603. H J, P PG, C BJ, R P, S BW, 1992. Presence of growth factors in human pituitary. Lab Inv 66: 639–645. H GN, H BL, S B, 1988. Diversity of prolactin systems in the insect Leucophaea maderae: use of antiserum polyclonality for immunocytochemical detection of neuropeptide heterogeneity. Cell Tissue Res 252: 557–563. H F, N HP, G CJP, 1997. Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the ThyroidStimulating Hormone, Follicle-Stimulating Hormone, Luteinizing Hormone/Choriogonadotropin receptor family from mammals. J Biol Chem 272: 1002–1010. H KY, T MO, K RJ, L SK, S YN, J ML, L DA, E WS, 1988. Effects of gonadal steroids on somatotroph function in the rat: analysis by the reverse hemolytic plaque assay. Endocrinology 123: 1405–1411. J L, Q X, K E, S N, S BW, K K, Y WF, L RV, 1997. Transforming growth Factor-, Transforming growth Factor- receptor I, and p27Kip1 expression in nontumorous and neoplastic human pituitaries. Am J Pathol 151: 509–519. K M, K I, O E, 1988. Activin-A: A modulator of multiple types of anterior pituitary cells. Biochem Biophys Res Commun 157: 48–54. K D, S D, F A, O E, 1998. PDGF and TGF- induce cell shape changes in invertebrates immunocytes via specific cell surface receptors. Eur J Cell Biol 75: 362–366.
891
K G, K RK, S W, D T, J C, W B, 1998. Identification of a new member of Transforming Growth Factor-Beta superfamily in Drosophila: the first invertebrate activin gene. Biochem Biophy Res Comm 246: 644–649. L OH, R NJ, F AL, R RJ, 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275. M J, 1990. The Transforming Growth Factor- family. Annu Rev Cell Biol 6: 597–641. M T, Y S, 1991. Transforming Growth Factor- and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system. Proc Soc Exp Biol Med 198: 599–605. M MA, H FM, 1991. Homologs of vertebrate growth factors in Drosophila melanogaster and other invertebrates. Curr Tops Dev Biol 24: 289–327. O MC, M HP, T M, BC L, 1999. Chronic effect of antidopaminergic drugs or estrogen on male Wistar rat lactotrophs and somatotrophs. Braz J Med Biol Res 32: 1561–1564. O E, F A, K D, B M, G S, 1997a. Involvement of PDGF and TGF- in cell migration and phagocytosis in invertebrate and human immunocytes. Anim Biol 6: 91–95. O E, S D, K D, 1997b. PDGF- and TGF-induced changes in cell shape of invertebrate immunocytes: effect of calcium entry blockers. Eur J Cell Biol 74: 336–341. P RW, S J RD, G WM, 1987. A transcript from Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature 325: 81–84. P M, D A, B N, V W, S DK, 1995. Reduction in the expression and action of transforming growth factor beta 1 on lactotropes during estrogeninduced tumorigenesis in the anterior pituitary. Cancer Res 55: 4892–4898. P A, S SB, H FM, 1997. Regulation of cell cycles synchronization by decapentaplegic during Drosophila eye development. Science 275: 203–206. R P, L C, J R, A PS, P D, R DL, 1996. Control of C. elegans larval development by neuronal expression of a TGF- homologue. Science 274: 1389–1391. R JR, E L JM, 1990. Inhibiting effect of mosquito larvae extract on the mitotic rate of hepatocytes of young growing and adult partially hepatectomized mice. Com Biol 9: 133–142. R JR, S AO, A CA, E L JM, 1994a. Inhibition of mouse parenchymal cells proliferation by mosquito larvae extract. Cell Mol Biol 40: 1183– 1188. R JR, O A, S A, E L JM, 1994b. Further action of mosquito larvae extract on the mitotic rate of mouse cell populations. BioCell 18: 51–57. R JR, 1996. Early effect of the mosquito larvae extract on the mouse cells proliferation. Cell Biol Int 20: 249–254. R JR, R FL, O AC, 1996. Mosquito larvae extract induces mitotic rate alterations of mice tongue keratinocytes along a circadian rhythm period. Cell Biol Int 20: 567–572. R JR, R OJ, S MA, B RR, 1999. Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation. Cell Biol Int 23: 219–226.
892
R JR, S MA, R OJ, F G, 2000. Partial characterisation of mosquito larvae extract inducing DNA synthesis alterations on human mononuclear cells. Cell Biol Int 24: 175–181. S D, P M, D A, E M, M H, G MB, 1998. Role of Transforming Growth Factor (TGF)- Type I and TGF- type II receptors in the TGF-I-regulated gene expression in pituitary prolactinsecreting lactotropes. Endocrinology 139: 3620–3628. S KP, M V, O B, P EF, 1989. Immunocytochemical localization of prolactin-like antigenic determinants in the neuroendocrine system of the honeybee (Apis mellifica). Histochemistry 91: 469–472. S KP, M V, H C, P EF, 1990. Ultrastructural localization of prolactin-like antigenic determinants in neurosecretory cells in the brain of the honeybee (Apis mellifica). Horm Metab Res 22: 413–417.
Cell Biology International, Vol. 25, No. 9, 2001
S K, B JV, V P, D L A, 1990. Immunocytochemical localization of human growth hormone- and prolactin-like antigenic determinants in the insects, Locusta migratoria and Sarcophaga bullata. Comp Biochem Physiol A 95: 373–378. T X, F AL, P RW, 1994. The Drosophila saxophone gene: A serine-Threonine kinase receptor of the TGF-beta superfamily. Science 263: 1756–1759. V JA, R-P HM, S H, P JM, 1985. Immunocytochemical localization of peptidergic neuron and neurosecretory cells in the neuroendocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides. Histochemistry 82: 9–18. W TK, M JP, 1995. Inhibin, activin and the female reproductive axis. Ann Rev Physiol 57: 219–224.