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Control of ovarian steroidogenesis in insects: A locust neurohormone is active in vitro on blowfly ovaries
General and Comparative Endocrinology 163 (2009) 292–297
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Control of ovarian steroidogenesis in insects: A locust neurohormone is active in vitro on blowfly ovaries G. Manière a, E. Vanhems b, I. Rondot c, J.P. Delbecque c,* a
Université de Bourgogne, CNRS, UMR 5548, 6 Boulevard Gabriel, F-21000 Dijon, France Université de Bordeaux, CNRS, France c Université de Bordeaux, CNRS, UMR 5228, Centre de Neurosciences Intégratives et Cognitives (CNIC), Avenue des Facultés, F-33405 Talence Cedex, France b
a r t i c l e
i n f o
Article history: Received 11 March 2009 Revised 10 April 2009 Accepted 27 April 2009 Available online 20 May 2009 Keywords: Insect reproduction Vitellogenesis Ecdysone Insulin-like peptides Bombyxin Neuroparsin
a b s t r a c t Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogenesis in blowfly isolated previtellogenic ovaries. However, such an activity was observed neither with authenticated neuroparsins (NPs), the putative homologs of the ovarian ecdysteroidogenic hormone of mosquitoes, nor with ovarian maturing peptide (OMP), the putative locust steroidogenic neurohormone. Partial purifications of CC extracts were then performed using methanol and/or acidic ethanol extractions followed by reverse phase HPLC and collected fractions were assayed in vitro. A significant steroidogenic activity was found in a single group of acidic fractions, well separated from OMP and NPs, which was associated to slight but significant anti-insulin immunoreactivity. In conclusion, a locust CC neurohormone, different from NPs and OMP, is able to stimulate ecdysteroidogenesis in blowfly ovaries. Though this active factor has not been fully characterized, its behavior during extraction or HPLC and its immunoreactivity strongly suggest it could be an insulin-like peptide. This is in agreement with previous studies demonstrating the role of such peptides as steroidogenic gonadotropins in blowflies and several other insects. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Steroidogenesis in insect ovaries is mainly controlled by stimulatory neuropeptides that are elaborated in the brain and secreted by the corpora cardiaca (Charlet et al., 1979; Hagedorn et al., 1979; see also the recent review by Brown et al., 2009). Our previous studies in Phormia regina have confirmed this general scheme by showing that blowfly brain contains at least two different factors, the main one present in the median neurosecretory cells of pars intercerebralis (PI) and acting on the ovary through the insulin signaling cascade (Manière et al., 2004), the second one, absent from PI, but present in the rest of the brain, acting through the cAMP signaling pathway (Manière et al., 2000). However, these factors have not yet been definitely identified in flies, despite attempts to extract and purify them (Adams et al., 1997, in Musca domestica and our unpublished studies in P. regina) and despite the considerable information obtained from the knowledge of Drosophila genome. * Corresponding author. E-mail address: [email protected] (J.P. Delbecque). 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.04.034
By contrast, Locusta migratoria is the insect species in which most of (if not all) the major peptides involved in the control of female reproduction have been identified, although information about their exact roles and about their probable interactions remains very incomplete. In particular, several investigations have enabled the identification of the three major brain neurohormones that are stored in the neurohemal lobes of corpora cardiaca (NCC), an organ having a very large size in locusts: (i) The first peptide family that has been identified in locust NCC is represented by neuroparsins (NPs, Girardie et al., 1987). Though NPs have been formerly described as antigonadotropic hormones in locusts, they present obvious sequence homologies with the ovarian ecdysteroidogenic hormone (OEH) isolated in Aedes aegypti (Brown et al., 1998; 2009), raising the question of their possible steroidogenic role in locusts, as well as in other insect species. (ii) The second major peptide isolated in NCC has been named Locusta insulin related protein (LIRP, Hétru et al., 1991). Although the physiological role of LIRP is not yet known in
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locusts, bombyxin, its probable homologue in Bombyx mori, has been described as a potent modulator of ecdysone biosynthesis in the molting glands of various lepidopteran larvae (Ishizaki and Suzuki, 1988; Nagata et al., 1999), as well as a putative regulator of oogenesis (reviewed in Brown et al., 2009; Claeys et al., 2002). More particularly, a synthetic bombyxin has been able in vitro to stimulate ovarian steroidogenesis in the blowfly (Manière et al., 2004) and a synthetic endogenous insulin-like peptide also has the same effect in the mosquito (Brown et al., 2008). (iii) Yet, despite the presence in locusts of two major peptides related to ecdysteroidogenic hormones in other insects, a third major NCC peptide, ovarian maturing parsin (OMP, Girardie and Girardie, 1996), has been proposed to be the putative ecdysteroidogenic gonadotropin of locusts, because it is able to increase ecdysteroid levels in female locusts in vivo (Girardie et al., 1998a). Thus, adult locust NCC represents an abundant source of three potential ecdysteroidogenic neurohormones. In order to examine whether these peptides could have a steroidogenic activity in a distant insect, we investigated their in vitro effects on the previtellogenic ovaries of our model, the blowfly P. regina.
2. Materials and methods 2.1. Insects Last instar larvae of P. regina were purchased from La Verminière de l’Ouest (Tremblay, France) and maintained without food in controlled conditions (25 °C, 16 h light/8 h darkness cycle), as previously described (Manière et al., 2000). After metamorphosis, newly ecdysed flies were supplied with sugar and water, but without any protein source, in order to prevent oocyte growth and vitellogenesis in females. Flies used in this study were thus only previtellogenic females. L. migratoria eggs were obtained from Prof. D. J. Van der Horst (Utrecht University, The Netherlands). Larvae and adults were reared in the gregarious phase as described by Girardie, 1966). After anesthesia with CO2, experimental animals were rapidly decapitated and placed on crushed ice. Dissections were made under a dissecting microscope, in dry conditions. 2.2. In vitro experiments Blowfly ovaries, extirpated under sterile conditions from 5 to 7 day old previtellogenic females, were rinsed 4 times with Grace’s insect culture medium (Gibco, Life Technologies-France, CergyPontoise), placed separately in 50 ll culture medium into sterile 96-well plates and incubated overnight at 26 ± 1 °C. Experiments were performed as previously described (Manière et al., 2000): in general rule, one ovary from an individual pair was incubated in the presence of a neurohormonal extract or a purified peptide (see below), whereas the contralateral ovary was used as its corresponding untreated control. Ovarian secretions of ecdysteroids into the culture medium were then compared by immunoassay (see below). 2.3. Peptides NPs, previously prepared under two highly purified forms corresponding to NP-A and NP-B (Girardie et al., 1989), were given by Dr. J. Girardie (University of Bordeaux). Synthetic OMP (Girardie et al., 1991) was also a gift from Dr. J. Girardie.
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2.4. NCC extracts and high performance liquid chromatography In order to prepare NCC extracts, adult female locusts (whatever their age) were anesthetized and dissected. Their NCC were removed, washed and pooled either in culture medium or in pure methanol (depending on the following treatment), then stored at 20 °C, over a short period. Pools of ca. one hundred NCC were then submitted to different extraction protocols: (a) ‘‘crude” extracts were made by homogenization of a NCC pool in Grace’s insect culture medium, followed by filtration on a 0.22 lm membrane; (b) methanolic extracts were made in cold conditions by homogenizing a NCC pool, first in pure methanol and then in aqueous methanol (70% methanol); (c) acidic extracts were made by homogenizing either a NCC pool or the pellets from the previous methanolic extracts, in cold 75% ethanol containing 0.2 M HCl (according to the protocol described by Hétru et al., 1991). Each treatment was followed by centrifugation (30,000g, 20 min, 2 °C), after which the pellet was re-extracted and centrifuged again; then the two corresponding supernatants were pooled. When necessary, solvents were dried using a Speed-Vac apparatus. High performance liquid chromatography (HPLC) was performed using a Beckmann Gold system, equipped with a multichannel UV detector, on a C8 reverse-phase column (Vydac, 250 mm length 4.6 mm i.d., 5 lm particles, 300 Å pores), with a 1 ml/min flow rate and a linear gradient from 18% to 55% acetonitrile in water containing 0.1% TFA (by volumes) during 36 min, followed by a wash with 65% acetonitrile (+0.1% TFA) during 20 min. Fractions were collected every min. 2.5. Immunoassays Ecdysteroids: they were measured as previously described (Manière et al., 2000), with an enzyme immunoassay, derived from that of Porcheron et al. (1989), but using the polyclonal L2 antiecdysone antibody and a 20-hydroxyecdysone-peroxidase tracer (Pascual et al., 1995). Measurements were generally made from the medium incubated with a single ovary, diluted in phosphate buffer (0.1 M, pH 7.4). In some cases however, measurements were also performed from ovaries, after classical extraction by methanol, in order to check their ecdysteroid contents either before or after culture (‘‘before” being done on similar ovaries taken in the same series than those really incubated). The amounts of hormones were determined by comparison with dose–response curves using ecdysone as a standard, this compound being the main ecdysteroid secreted by blowfly ovaries. Consequently, the data were calculated as pg ecdysone-equivalents per culture. The lower detection limit of our assay was about 1 pg ecdysone per well (but about 7–10 pg per ovary, due to dilution of the incubation medium in phosphate buffer). However, in order to minimize the variations due to the size and age of flies, as well as to variations in the incubation time (‘‘overnight” incubations varying from 12 to 18 h), a ratio was calculated for each individual experiment, with the concentration of ecdysteroids in the treated ovary, in the numerator, over that of its control (i.e., contra-lateral) ovary, in the denominator (see Manière et al., 2000, for more details). The treated/control ratios from different animals were then averaged and the data presented as relative means (±S.E.M.). Statistical analysis was generally made using Student’s t-tests by comparing concentration ratios to the theoretical value of 1 (which is equivalent to a paired t-test). Insulin-like peptides: following similar enzyme immunoassay protocol, detection of insulin-like material was attempted, using a bovine insulin labelled with peroxidase as tracer (purchased from Sigma–Aldrich) and a specific anti-insulin antibody (given by Prof. R. Moreau, Bordeaux, France; see Bounias et al., 1993). Because of the unknown affinity of this antibody for insect insulin-like pep-
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tides and the lack of purified LIRP, no calibration was conceivable, but the presence or absence of such peptides were observable, by changes in the binding of peroxidase tracer. 3. Results 3.1. Effects of locust NCC crude extracts on blowfly ovarian steroidogenesis Our previous studies have shown that blowfly ovaries from a same pair, isolated from 5 to 7 day-old previtellogenic females, can be stimulated to synthesize ecdysteroids and to secrete them in very similar quantities, without storing them, giving the possibility of a sensitive bioassay for factors regulating steroidogenesis, either positively (Manière et al., 2000) or negatively (Manière et al., 2002, 2003). Crude extracts from Locusta NCC were thus tested in this bioassay, after verification that they did not contain detectable amounts of immunoreactive ecdysteroids in the volume used for in vitro incubations (<10 pg/50 ll culture medium, n = 5). Experiments were performed as follows: from each previtellogenic female, one of the two ovaries was incubated in the medium containing a NCC extract (treated ovary), the contralateral ovary being incubated in pure medium (control ovary). Then, ecdysteroids secreted into the incubation medium by each ovary were measured using enzyme immunoassay. Results, given in Fig. 1 show that presence of extracts corresponding to 2 NCC in Grace’s medium stimulated previtellogenic ovaries to produce significantly higher amounts of ecdysteroids than their respective contralateral ovaries. We have also verified that blowfly ovaries, extracted either before or after in vitro incubations, contained no detectable amount of immunoreactive compounds (<10 pg E-eq./ovary, n = 5 for both cases). Thus, we can assume that ecdysteroids secreted in the culture media after stimulation by NCC extracts were the result of active biosynthesis. Results given in Fig. 2, expressed as the means of individual treated/control ratios, show that ecdysteroidogenic activity varied approximately as a linear function of NCC concentration, within the range tested. Because such ratios are more accurate to compare from an experiment to another and as they also minimize variations between individuals, they will be preferred in the following experiments.
8 means of treated /control r atios
294
7 6 5 4 3 2 1 0 0
1
2 3 NCC equivalents
Fig. 2. Ecdysteroidogenic activity in vitro expressed as means (±SEM, n = 4–10 per point) of individual treated/control ratios in function of NCC equivalents. Points over 1 NCC equiv. were significantly different from their respective controls. Linear regression indicated that activity could be estimated as: y = 2.1 NCC + 1.0 (r2 = 0.98).
3.2. Effect of authenticated peptides on ovarian steroidogenesis Experiments using the same in vitro test were then performed with authenticated locust neuropeptides, namely NPs, obtained from Dr. J. Girardie in two highly purified forms, NP-A and NP-B, and OMP, available in synthetic form. Results, summarized in Table 1, showed that none of these peptides had a significant steroidogenic effect on blowfly ovaries at similar concentrations than those previously tested for crude extracts. In the experiments shown in Table 1, for better comparison with our other experiments, OMP concentrations were expressed in NCC equivalents, using the approximate content of OMP in NCC measured by Dr. J Girardie (personal communication). However, as this peptide was available in synthetic form, its activity has also been tested in molar concentrations ranging from 10 9 M to 10 5 M (by a step 10): no steroidogenic activity was observed during these experiments, apart from a slight but significant activity at the highest concentration (stimulation ratio of 1.9 ± 0.3, n = 10, at 10 5 M, corresponding approximately to 25 NCC equiv. per culture), which, however, was not comparable to the high activity of NCC extracts.
200
pg ecdysone equivalents
3.3. Partial purification of NCC extracts Partial purification of NCC extracts was then undertaken in order to better identify the steroidogenic factor(s) responsible for the stimulation of blowfly ovaries. A simple analysis of extraction procedures was performed using three different solvent conditions,
100
0
ovaries before culture
0 NCC (controls)
2 NCC (treated)
ovaries after culture
Fig. 1. In vitro effects of crude NCC extracts on ovarian steroidogenesis by blowfly isolated ovaries. Ecdysteroids expressed as pg of ecdysone equivalents per ovary or per culture were measured (i) in the ovaries (n = 5) either before or after culture (‘‘before” being done on similar ovaries than those really incubated) and (ii) in the culture media after overnight incubation of ovaries (n = 10) in the absence (controls, 0 NCC) or in the presence (2 NCC equivalents) of crude NCC extracts. Mean secretion (±SEM, error bars) of ecdysteroids after stimulation by 2 NCC was significantly different from that of controls (Student t-test, P < 0.05). Values extracted in the ovaries were in both cases at the background sensitivity of the method.
Table 1 Effect of highly purified NPs (NP-A and NP-B) and of synthetic OMP on ovarian steroidogenesis in blowflies. In the case of OMP, 1 NCC-equivalent is roughly supposed to correspond to 2 ng/ll for a total volume of 50 ll incubation medium and represents ca. 4.10 7 M (personal communication from Dr. J. Girardie). None of the ratios was significantly different from the theoretical value of 1 (statistics using Student’s t-test, P < 0.05). Neuropeptides
Neuropeptide amounts in NCC equivalents
n
Results expressed as treated/control ratios
Purified NP-A Purified NP-A Purified NP-B Purified NP-B Synthetic OMP Synthetic OMP
2 5 2 5 2 (4 ng/ll) 5 (10 ng/ll)
11 7 10 10 9 20
1.2 ± 0.2 1.1 ± 0.2 1.1 ± 0.2 1.4 ± 0.2 1.3 ± 0.2 1.4 ± 0.2
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G. Manière et al. / General and Comparative Endocrinology 163 (2009) 292–297 Table 2 Steroidogenic activity of locust NCC extracts (2 NCC-equiv. per culture), after successive extractions using various solvent conditions. (): significantly different from the theoretical value of 1 (Student’s t-test, P < 0.05).
applied successively to the same NCC pools. Results presented in Table 2 show that, though some steroidogenic activity was partly soluble in 100% and 70% methanol, the main part of this activity was only soluble after an acidic ethanol extraction. Extraction in pure methanol, known to be favorable to purification of NPs and OMP (Girardie et al., 1989; 1991; 1998b), did not allowed a sufficient recovery of ecdysteroidogenic activity, confirming our above results with authenticated peptides. 3.4. HPLC of NCC peptides
NPs 0.5
OMP
1.7 ± 0.1 () 2.8 ± 0.3 () 4.4 ± 0.9 ()
0 1
B
NPs OMP
Treated/control ratios
8 8 8
absorbance
n
100% methanol 70% methanol Acidic ethanol
A
absorbance
Extraction conditions
1
0.5
CP
LIRP
3.5. Steroidogenic activity of HPLC fractions Analysis of the HPLC fractions obtained from acidic ethanol extracts, in terms of their capacity to modify ovarian ecdysteroidogenesis in our in vitro test, revealed that a high steroidogenic activity was found in a single group of fractions co-migrating with the UV-peak tentatively identified as LIRP and reached a maximum at fraction #21 that showed anti-insulin immunoreactivity (Fig. 3C). No significant activity, neither steroidogenic nor steroidostatic (i.e., inhibitory), was found in the other fractions, in particular those corresponding to NPs and OMP, confirming the observations made with authenticated peptides at the same concentrations. Incidentally, the peak corresponding to the putative C-peptide was not significantly active. Importantly, we also observed the same pattern of activity after methanol extraction
0 steroidogenic activity
Analysis of NCC extracts was then made by HPLC, using a reverse-phase procedure adapted from that described by Hétru et al. (1991), in order to facilitate the identification of the main peaks displayed by UV detector. Interestingly, UV-peaks corresponding to OMP and NPs (identified by comparison with the retention time of reference compounds) were observed whatever the extraction procedure: indeed, these neurohormones were satisfactorily recovered after methanol extraction (Fig. 3A), as well as after acidic ethanol extraction (Fig. 3B). In this second case however, OMP and NPs were accompanied by other UV-peaks, in particular by a peak which, as judged from its relative retention time, behave chromatographically very similarly as LIRP, previously identified by Hétru et al. (1991). Indeed, this peak migrated before OMP and NPs, at ca. 80% of NP retention time in similar HPLC conditions. In addition, the abundance of this peak in acidic conditions, which was the major component of NCC after NPs, and its absence from the chromatogram obtained with methanolic extracts, were in agreement with the observations of Hétru et al. (1991) and corroborated its identification as LIRP. Moreover, the HPLC fraction #21 containing most of this peak showed a low but significant affinity for anti-insulin polyclonal antibodies, confirming the presence of an insulin-like peptide in this fraction. Incidentally, acidic extraction also enabled the observation of smaller peaks, migrating before putative LIRP, possibly corresponding to truncated LIRP forms observed by Hétru et al. (1991), and of a peak with a higher retention time than NPs, probably corresponding to the C-peptide (the peptide resulting from the cleavage of A and B chains from insulin precursor), also identified by Hétru et al. (1991).
3
C 2
*
1 0
20
40
time (min) 60
Fig. 3. HPLC separation of NCC neuropeptides after (A) methanol extraction or (B) acidic ethanol extraction. Ordinate indicates absorbance at 225 nm. Abscissa indicates time in min. Retention times of OMP and NPs were confirmed by similar HPLCs made with authenticated peptides. Peaks supposed to correspond to LIRP or C-peptide (CP) were tentatively identified on the basis of the data of Hétru et al. (1991). (C) Steroidogenic activity of the fractions (1 fraction per min) corresponding to 2 NCC equivalents, separated after acidic ethanol extraction. Results (ordinate: steroidogenic activity) are expressed as treated/control ratios. As no steroidostatic activity was found, ordinate below 1 is useless. Fractions 1–17 and 31–60 are not shown (they have not been assayed one by one, but by pools that were found inactive). Errors bars indicate means ± SEM obtained from 5 to 6 EIA measurements. Points over 1.5 were significantly different from their respective controls. Asterisk indicates a fraction with slight anti-insulin immunoreactivity.
(not shown), but at a much lower level, suggesting that the lower activity obtained after this extraction protocol was only due to a lower recovery of the same active factor. A few other experiments (not shown) were also done by pooling different HPLC fractions, in particular those corresponding to LIRP plus OMP and NPs, which did not reveal supplementary activity, suggesting that possible interactions between these different peptides were not beneficial for steroidogenesis in isolated blowfly ovaries. Altogether, HPLC results confirmed that neither OMP nor NPs were able to explain the high steroidogenic activity of NCC extracts in blowflies, but demonstrated that another (and probably single) factor, migrating like LIRP, was responsible for this effect. 4. Discussion 4.1. Activity of locust NCC crude extracts on blowfly ovaries Our results first show that extracts from locust NCC are able to trigger ovarian secretion of ecdysteroids in an insect, the blowfly,
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belonging to a very distant group. This secretion is obviously due to an increase in steroidogenesis, as blowfly previtellogenic ovaries do not store significant amounts of immunoreactive ecdysteroids. The precise dissection of locust large sized NCC, washed from hemolymph contamination, and the use of various extraction procedures also confirm that biological activity was likely associated with neuropeptides. Indeed, our results might not be attributed to the artefactual presence of some ecdysteroids in NCC extracts, as indicated by the absence of immunoreactive ecdysteroids in crude extracts. More importantly, even the transformation of non-immunoreactive ecdysteroids, such as putative precursors or conjugates, into immunoreactive ones can be ruled out in our extracts. Indeed, during the various steps of NCC purification, the main part of steroidogenic activity remained in the pellets and was badly extracted by successive treatments with pure and with 70% methanol, each of them being yet considered very suitable for dissolving most steroid compounds. By contrast, activity was efficiently extracted from such pellets by acidic ethanol, a treatment that is suitable for many peptides, but that is not particularly recommended for steroids. Our HPLC results also indicate that only one group of fractions was active on blowfly ovaries. This observation is relatively surprising, as our previous studies have shown that ecdysteroidogenesis in blowfly ovaries is modulated by at least two different stimulatory signaling pathways (Manière et al., 2000), as well as by two different inhibitory ones (Manière et al., 2002, 2003). It can thus be assumed that only one of the blowfly factors has a homologue in locust NCC and that the others either have diverged too much during evolution or are produced by other site(s) of secretion. 4.2. Locust NPs and OMP are not active on blowfly ovaries Our data also indicate that NPs (either purified NP-A and NP-B prepared by Dr. J. Girardie or our own HPLC fractions co-migrating as NPs) have no significant ecdysteroidogenic effect on blowfly ovaries. Though this finding appears consistent with the antigonadotropic role of NPs previously described in locusts (Girardie et al., 1987), it is yet surprising because NPs also have some sequence similarities with OEH, the steroidogenic hormone of female mosquitoes (Brown et al., 1998). It is thus possible that NPs, OEH and their putative homologues in flies have diverged too much during evolution to bind with the same ligands or with the same receptors and to fulfill the same functions. It is also possible that NPs, which also display similarities with vertebrate insulin-like growth factor binding proteins (Badisco et al., 2007; Claeys et al., 2003) and interact in vitro with insulin-like peptides (Badisco et al., 2008), are unable to exert their action alone in blowflies, but the lack of a significant effect when NPs were pooled with LIRP containing fractions in our conditions remains difficult to interpret. However, another possibility could be that NP/OEH homologues are absent in flies and have no physiological role, as suggested by the observation that no corresponding gene has been identified so far in Drosophila genome (Hewes and Taghert, 2001; Nässel, 2002), although similar sequences have been found in various other invertebrates (Claeys et al., 2003). Our results also showed that OMP is not the locust NCC factor able to trigger acute ecdysteroidogenesis in blowfly ovaries. Indeed, though OMP seemed to have a slight ecdysteroidogenic effect at a very high concentration in our in vitro tests on blowfly ovaries, it was inactive at the concentrations at which crude extracts were already very efficient. However, if a peptide homologue of OMP would exist in blowflies, it seems highly probable that it should be active at lower concentrations. OMP has been described as a putative gonadotropin in Locusta and in Schistocerca (Girardie and Girardie, 1996; Girardie et al., 1998b) on the basis of several
in vivo evidences, but its capacity to stimulate ovarian steroidogenesis has not yet been confirmed in vitro, i.e., directly on locust ovaries. Moreover, the presence of OMP seems to be restricted to Acridae (Richard et al., 1994) and homologues or orthologues have not been described in other insects so far. Thus, although our present data do not discard the possibility for OMP to be involved in the control of ovarian ecdysteroidogenesis in locusts, they clearly show that this neuropeptide, either alone or pooled with other NCC fractions, is not responsible for the steroidogenic effect of locust NCC extracts on blowfly ovaries. 4.3. Gonadotropic role of insulin-like peptides The main positive data obtained during this study indicate that the steroidogenic factor active on blowfly ovaries has several properties suggesting it may be LIRP, the only insulin-like protein present in locust NCC according to Hétru et al. (1991). Indeed, the steroidogenic activity is mainly recovered after extraction in acidic conditions, but not after neutral ones, as it is generally the case for insulin-like proteins (Teller and Pilc, 1985), including LIRP (Hétru et al., 1991). Moreover, the only group of HPLC fractions having a steroidogenic activity clearly migrated like a very high UV peak, corresponding, in terms of its relative abundance and retention time, to a compound previously identified as LIRP by Hétru et al. (1991) in a similar separation system. Lastly, anti-insulin-like immunoreactivity was found in the HPLC fraction having the highest steroidogenic activity. Since NCC contain only three major neurohormones and since the steroidogenic factor is neither OMP nor NPs, then the possibility that it could be the third one, i.e., LIRP, appears very likely. By contrast, the possibility that the steroidogenic factor could be a minor compound migrating in the same fractions as LIRP, although not definitively ruled out by our experiments, appears less credible. Unfortunately, we have been compelled to interrupt this work on insect ovarian steroidogenesis before the complete identification of the active factor. Further studies in other laboratories will be necessary to obtain a definitive proof, requiring the complete purification or the chemical synthesis of LIRP and the verification of its activity in similar bioassays. Nevertheless, our present experimental data are strongly in agreement with our previous studies having already shown that exogenous insulin-like peptides from distant animals, namely bovine insulin and bombyxin II, as well as endogenous insulin-like peptides from medial neurosecretory cells, are able to stimulate ecdysteroidogenesis in blowfly isolated ovaries (Manière et al., 2004). Moreover, they are also in good agreement with several observations in some other insects suggesting that insulin-like peptides have a steroidogenic role during oogenesis (reviewed in Claeys et al., 2002; Garofalo, 2002; Brown et al., 2009), as well as during post-embryonic development. In conclusion, a peptide from locust NCC has a steroidogenic activity on blowfly ovaries. Though this factor has not been definitely identified, the present results show that it is neither NPs nor OMP, but probably LIRP, in agreement with our previous observations showing that insulin-like peptides are major steroidogenic gonadotropins in blowflies. Further studies are needed to verify whether this property of insulin-like peptides, only observed in mosquitoes and flies (review in Brown et al., 2009), can be generalized to other insect groups and, more particularly, whether LIRP also has such a function in locusts. Acknowledgments The authors thank Josiane and Adrien Girardie (now retired) for their help and support during this study. They also thank M. De Reggi (Marseille), for the gift of anti-ecdysone antibodies.
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References Adams, T.S., Gerst, J.W., Masler, E.P., 1997. Regulation of ovarian ecdysteroid production in the housefly, Musca domestica. Arch. Insect Biochem. Physiol. 35, 135–148. Badisco, L., Claeys, I., Van Loy, T., Van Hiel, M., Franssens, V., Simonet, G., Vanden Broeck, J., 2007. Neuroparsins, a family of conserved arthropod neuropeptides. Gen. Comp. Endocrinol. 153, 64–71. Badisco, L., Claeys, I., Van Hiel, M., Clynen, E., Huybrechts, J., Vandersmissen, T., Van Soest, S., Vanden Bosch, L., Simonet, G., Vanden Broeck, J., 2008. Purification and characterization of an insulin-related peptide in the desert locust, Schistocerca gregaria: immunolocalization, cDNA cloning, transcript profiling and interaction with neuroparsin. J. Mol. Endocrinol. 40, 137–150. Bounias, M., Bahjou, A., Gourdoux, L., Moreau, R., 1993. Molecular activation of a trehalase purified from the fat body of a coleopteran insect (Tenebrio molitor), by an endogenous insulin-like peptide. Biochem. Mol. Biol. Int. 31, 249–266. Brown, M.R., Graf, R., Swiderek, K.M., Fendley, D., Strackert, T.H., Champagne, D.E., Lea, A.O., 1998. Identification of a steroidogenic neurohormone in female mosquitoes. J. Biol. Chem. 273, 3967–3971. Brown, M.R., Clark, K.D., Gulia, M., Zhao, Z., Garczynski, S.F., Crim, J.W., Suderman, R.J., Strand, M.R., 2008. An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti. Proc. Natl. Acad. Sci. USA 105, 5716– 5721. Brown, M.R., Sieglaff, D.H., Rees, H.H., 2009. Gonadal ecdysteroidogenesis in Arthropoda: occurrence and regulation. Ann. Rev. Entomol. 54, 105–125. Charlet, M., Goltzene, F., Hoffmann, J.A., 1979. Experimental evidence for a neuroendocrine control of ecdysone biosynthesis in adult females of Locusta migratoria. J. Insect Physiol. 25, 463–466. Claeys, I., Simonet, G., Poels, J., Van Loy, T., Vercammen, L., De Loof, A., Vanden Broeck, J., 2002. Insulin-related peptides and their conserved signal transduction pathway. Peptides 23, 807–816. Claeys, I., Simonet, G., Van Loy, T., De Loof, A., Vanden Broeck, J., 2003. cDNA cloning and transcript distribution of two novel members of the neuroparsin family in the desert locust, Schistocerca gregaria. Insect Mol. Biol. 12, 473–481. Garofalo, R.S., 2002. Genetic analysis of insulin signaling in Drosophila. Trends Endocrinol. Metabol. 13, 156–162. Girardie, A., 1966. Contrôle de l’activité génitale chez Locusta migratoria Mise en évidence d’un facteur gonadotrope et d’un facteur allatotrope dans la pars intercerebralis. Bull. Soc. Zool. France 91, 423–439. Girardie, J., Girardie, A., 1996. Lom OMP, a putative ecdysiotropic factor for the ovary in Locusta migratoria. J. Insect Physiol. 42, 215–221. Girardie, J., Boureme, D., Couillaud, F., Tamarelle, M., Girardie, A., 1987. Antijuvenile effect of neuroparsin A, a neuroprotein isolated from locust corpora cardiaca. Insect Biochem., 977–984. Girardie, J., Girardie, A., Huet, J.C., Pernollet, J.C., 1989. Amino acid sequence of locust neuroparsins. FEBS Lett. 245, 4–8. Girardie, J., Richard, O., Huet, J.C., Nespoulous, C., Van Dorsselaer, A., Pernollet, J.C., 1991. Physical characterization and sequence identification of the Ovary
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Maturating Parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust (Locusta migratoria migratorioides). Eur. J. Biochem. 202, 1121–1126. Girardie, J., Geoffre, S., Delbecque, J.P., Girardie, A., 1998a. Arguments for two distinct gonadotropic activities triggered by different domains of the ovary maturating parsin of Locusta migratoria. J. Insect Physiol. 44, 1063–1071. Girardie, J., Huet, J.C., Atay-Kadiri, Z., Ettaouil, S., Delbecque, J.P., Fournier, B., Pernollet, J.C., Girardie, A., 1998b. Isolation, sequence determination, physical and physiological characterization of the neuroparsins and ovary maturing parsins of Schistocerca gregaria. Insect Biochem. Mol. Biol. 28, 641–650. Hagedorn, H.H., Shapiro, J.P., Hanaoka, K., 1979. Ovarian ecdysone secretion is controlled by a brain hormone in an adult mosquito. Nature 282, 92–94. Hétru, C., Li, K.W., Bulet, P., Lagueux, M., Hoffmann, J.A., 1991. Isolation and structural characterization of an insulin-related molecule, a predominant neuropeptide from Locusta migratoria. Eur. J. Biochem. 201, 495–499. Hewes, R.S., Taghert, P.H., 2001. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res. 11, 1126–1142. Ishizaki, H., Suzuki, A., 1988. An insect brain peptide as a member of insulin family. Horm. Metab. Res. 20, 426–429. Manière, G., Vanhems, E., Delbecque, J.P., 2000. Cyclic AMP-dependent and independent stimulations of ovarian steroidogenesis by brain factors in the blowfly, Phormia regina. Mol. Cell Endocrinol. 168, 31–40. Manière, G., Vanhems, E., Gautron, F., Delbecque, J.P., 2002. Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina. J. Endocrinol. 173, 533–544. Manière, G., Vanhems, E., Gautron, F., Delbecque, J.P., 2003. Inhibition of ovarian steroidogenesis by cyclic-GMP in a fly. J. Endocrinol. 177, 35–44. Manière, G., Rondot, I., Büllesbach, E.E., Gautron, F., Vanhems, E., Delbecque, J.P., 2004. Control of ovarian steroidogenesis by insulin-like peptides in the blowfly (Phormia regina). J. Endocrinol. 181, 147–156. Nagata, K., Maruyama, K., Kojima, K., Yamamoto, M., Tanaka, M., Kataoka, H., Nagasawa, H., Isogai, A., Ishizaki, H., Suzuki, A., 1999. Prothoracicotropic activity of SBRPs, the insulin-like peptides of the saturniid silkworm Samia cynthia ricini. Biochem. Biophys. Res. Commun. 266, 575–578. Nässel, D.R., 2002. Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog. Neurobiol. 68, 1–84. Pascual, N., Bellés, X., Delbecque, J.P., Hua, Y.J., Koolman, J., 1995. Quantification of ecdysteroids by immunoassay: comparison of enzyme immunoassay and radioimmunoassay. Z. Naturforsch. [C] 50, 862–867. Porcheron, P., Morinière, M., Grassi, J., Pradelles, P., 1989. Development of an enzyme immunoassay for ecdysteroids using acetylcholinesterase as label. Insect Biochem. 19, 117–122. Richard, O., Tamarelle, M., Geoffre, S., Girardie, J., 1994. Restricted occurrence of Locusta migratoria ovary maturing parsin in the brain–corpora cardiaca complex of various insect species. Histochemistry 102, 233–239. Teller, J.K., Pilc, L., 1985. Insulin in insects: analysis of immunoreactivity in tissue extracts. Comp. Biochem. Physiol. B 81, 493–497.
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Control of ovarian steroidogenesis in insects: A locust neurohormone is active in vitro on blowfly ovaries G. Manière a, E. Vanhems b, I. Rondot c, J.P. Delbecque c,* a
Université de Bourgogne, CNRS, UMR 5548, 6 Boulevard Gabriel, F-21000 Dijon, France Université de Bordeaux, CNRS, France c Université de Bordeaux, CNRS, UMR 5228, Centre de Neurosciences Intégratives et Cognitives (CNIC), Avenue des Facultés, F-33405 Talence Cedex, France b
a r t i c l e
i n f o
Article history: Received 11 March 2009 Revised 10 April 2009 Accepted 27 April 2009 Available online 20 May 2009 Keywords: Insect reproduction Vitellogenesis Ecdysone Insulin-like peptides Bombyxin Neuroparsin
a b s t r a c t Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogenesis in blowfly isolated previtellogenic ovaries. However, such an activity was observed neither with authenticated neuroparsins (NPs), the putative homologs of the ovarian ecdysteroidogenic hormone of mosquitoes, nor with ovarian maturing peptide (OMP), the putative locust steroidogenic neurohormone. Partial purifications of CC extracts were then performed using methanol and/or acidic ethanol extractions followed by reverse phase HPLC and collected fractions were assayed in vitro. A significant steroidogenic activity was found in a single group of acidic fractions, well separated from OMP and NPs, which was associated to slight but significant anti-insulin immunoreactivity. In conclusion, a locust CC neurohormone, different from NPs and OMP, is able to stimulate ecdysteroidogenesis in blowfly ovaries. Though this active factor has not been fully characterized, its behavior during extraction or HPLC and its immunoreactivity strongly suggest it could be an insulin-like peptide. This is in agreement with previous studies demonstrating the role of such peptides as steroidogenic gonadotropins in blowflies and several other insects. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Steroidogenesis in insect ovaries is mainly controlled by stimulatory neuropeptides that are elaborated in the brain and secreted by the corpora cardiaca (Charlet et al., 1979; Hagedorn et al., 1979; see also the recent review by Brown et al., 2009). Our previous studies in Phormia regina have confirmed this general scheme by showing that blowfly brain contains at least two different factors, the main one present in the median neurosecretory cells of pars intercerebralis (PI) and acting on the ovary through the insulin signaling cascade (Manière et al., 2004), the second one, absent from PI, but present in the rest of the brain, acting through the cAMP signaling pathway (Manière et al., 2000). However, these factors have not yet been definitely identified in flies, despite attempts to extract and purify them (Adams et al., 1997, in Musca domestica and our unpublished studies in P. regina) and despite the considerable information obtained from the knowledge of Drosophila genome. * Corresponding author. E-mail address: [email protected] (J.P. Delbecque). 0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.04.034
By contrast, Locusta migratoria is the insect species in which most of (if not all) the major peptides involved in the control of female reproduction have been identified, although information about their exact roles and about their probable interactions remains very incomplete. In particular, several investigations have enabled the identification of the three major brain neurohormones that are stored in the neurohemal lobes of corpora cardiaca (NCC), an organ having a very large size in locusts: (i) The first peptide family that has been identified in locust NCC is represented by neuroparsins (NPs, Girardie et al., 1987). Though NPs have been formerly described as antigonadotropic hormones in locusts, they present obvious sequence homologies with the ovarian ecdysteroidogenic hormone (OEH) isolated in Aedes aegypti (Brown et al., 1998; 2009), raising the question of their possible steroidogenic role in locusts, as well as in other insect species. (ii) The second major peptide isolated in NCC has been named Locusta insulin related protein (LIRP, Hétru et al., 1991). Although the physiological role of LIRP is not yet known in
G. Manière et al. / General and Comparative Endocrinology 163 (2009) 292–297
locusts, bombyxin, its probable homologue in Bombyx mori, has been described as a potent modulator of ecdysone biosynthesis in the molting glands of various lepidopteran larvae (Ishizaki and Suzuki, 1988; Nagata et al., 1999), as well as a putative regulator of oogenesis (reviewed in Brown et al., 2009; Claeys et al., 2002). More particularly, a synthetic bombyxin has been able in vitro to stimulate ovarian steroidogenesis in the blowfly (Manière et al., 2004) and a synthetic endogenous insulin-like peptide also has the same effect in the mosquito (Brown et al., 2008). (iii) Yet, despite the presence in locusts of two major peptides related to ecdysteroidogenic hormones in other insects, a third major NCC peptide, ovarian maturing parsin (OMP, Girardie and Girardie, 1996), has been proposed to be the putative ecdysteroidogenic gonadotropin of locusts, because it is able to increase ecdysteroid levels in female locusts in vivo (Girardie et al., 1998a). Thus, adult locust NCC represents an abundant source of three potential ecdysteroidogenic neurohormones. In order to examine whether these peptides could have a steroidogenic activity in a distant insect, we investigated their in vitro effects on the previtellogenic ovaries of our model, the blowfly P. regina.
2. Materials and methods 2.1. Insects Last instar larvae of P. regina were purchased from La Verminière de l’Ouest (Tremblay, France) and maintained without food in controlled conditions (25 °C, 16 h light/8 h darkness cycle), as previously described (Manière et al., 2000). After metamorphosis, newly ecdysed flies were supplied with sugar and water, but without any protein source, in order to prevent oocyte growth and vitellogenesis in females. Flies used in this study were thus only previtellogenic females. L. migratoria eggs were obtained from Prof. D. J. Van der Horst (Utrecht University, The Netherlands). Larvae and adults were reared in the gregarious phase as described by Girardie, 1966). After anesthesia with CO2, experimental animals were rapidly decapitated and placed on crushed ice. Dissections were made under a dissecting microscope, in dry conditions. 2.2. In vitro experiments Blowfly ovaries, extirpated under sterile conditions from 5 to 7 day old previtellogenic females, were rinsed 4 times with Grace’s insect culture medium (Gibco, Life Technologies-France, CergyPontoise), placed separately in 50 ll culture medium into sterile 96-well plates and incubated overnight at 26 ± 1 °C. Experiments were performed as previously described (Manière et al., 2000): in general rule, one ovary from an individual pair was incubated in the presence of a neurohormonal extract or a purified peptide (see below), whereas the contralateral ovary was used as its corresponding untreated control. Ovarian secretions of ecdysteroids into the culture medium were then compared by immunoassay (see below). 2.3. Peptides NPs, previously prepared under two highly purified forms corresponding to NP-A and NP-B (Girardie et al., 1989), were given by Dr. J. Girardie (University of Bordeaux). Synthetic OMP (Girardie et al., 1991) was also a gift from Dr. J. Girardie.
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2.4. NCC extracts and high performance liquid chromatography In order to prepare NCC extracts, adult female locusts (whatever their age) were anesthetized and dissected. Their NCC were removed, washed and pooled either in culture medium or in pure methanol (depending on the following treatment), then stored at 20 °C, over a short period. Pools of ca. one hundred NCC were then submitted to different extraction protocols: (a) ‘‘crude” extracts were made by homogenization of a NCC pool in Grace’s insect culture medium, followed by filtration on a 0.22 lm membrane; (b) methanolic extracts were made in cold conditions by homogenizing a NCC pool, first in pure methanol and then in aqueous methanol (70% methanol); (c) acidic extracts were made by homogenizing either a NCC pool or the pellets from the previous methanolic extracts, in cold 75% ethanol containing 0.2 M HCl (according to the protocol described by Hétru et al., 1991). Each treatment was followed by centrifugation (30,000g, 20 min, 2 °C), after which the pellet was re-extracted and centrifuged again; then the two corresponding supernatants were pooled. When necessary, solvents were dried using a Speed-Vac apparatus. High performance liquid chromatography (HPLC) was performed using a Beckmann Gold system, equipped with a multichannel UV detector, on a C8 reverse-phase column (Vydac, 250 mm length 4.6 mm i.d., 5 lm particles, 300 Å pores), with a 1 ml/min flow rate and a linear gradient from 18% to 55% acetonitrile in water containing 0.1% TFA (by volumes) during 36 min, followed by a wash with 65% acetonitrile (+0.1% TFA) during 20 min. Fractions were collected every min. 2.5. Immunoassays Ecdysteroids: they were measured as previously described (Manière et al., 2000), with an enzyme immunoassay, derived from that of Porcheron et al. (1989), but using the polyclonal L2 antiecdysone antibody and a 20-hydroxyecdysone-peroxidase tracer (Pascual et al., 1995). Measurements were generally made from the medium incubated with a single ovary, diluted in phosphate buffer (0.1 M, pH 7.4). In some cases however, measurements were also performed from ovaries, after classical extraction by methanol, in order to check their ecdysteroid contents either before or after culture (‘‘before” being done on similar ovaries taken in the same series than those really incubated). The amounts of hormones were determined by comparison with dose–response curves using ecdysone as a standard, this compound being the main ecdysteroid secreted by blowfly ovaries. Consequently, the data were calculated as pg ecdysone-equivalents per culture. The lower detection limit of our assay was about 1 pg ecdysone per well (but about 7–10 pg per ovary, due to dilution of the incubation medium in phosphate buffer). However, in order to minimize the variations due to the size and age of flies, as well as to variations in the incubation time (‘‘overnight” incubations varying from 12 to 18 h), a ratio was calculated for each individual experiment, with the concentration of ecdysteroids in the treated ovary, in the numerator, over that of its control (i.e., contra-lateral) ovary, in the denominator (see Manière et al., 2000, for more details). The treated/control ratios from different animals were then averaged and the data presented as relative means (±S.E.M.). Statistical analysis was generally made using Student’s t-tests by comparing concentration ratios to the theoretical value of 1 (which is equivalent to a paired t-test). Insulin-like peptides: following similar enzyme immunoassay protocol, detection of insulin-like material was attempted, using a bovine insulin labelled with peroxidase as tracer (purchased from Sigma–Aldrich) and a specific anti-insulin antibody (given by Prof. R. Moreau, Bordeaux, France; see Bounias et al., 1993). Because of the unknown affinity of this antibody for insect insulin-like pep-
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tides and the lack of purified LIRP, no calibration was conceivable, but the presence or absence of such peptides were observable, by changes in the binding of peroxidase tracer. 3. Results 3.1. Effects of locust NCC crude extracts on blowfly ovarian steroidogenesis Our previous studies have shown that blowfly ovaries from a same pair, isolated from 5 to 7 day-old previtellogenic females, can be stimulated to synthesize ecdysteroids and to secrete them in very similar quantities, without storing them, giving the possibility of a sensitive bioassay for factors regulating steroidogenesis, either positively (Manière et al., 2000) or negatively (Manière et al., 2002, 2003). Crude extracts from Locusta NCC were thus tested in this bioassay, after verification that they did not contain detectable amounts of immunoreactive ecdysteroids in the volume used for in vitro incubations (<10 pg/50 ll culture medium, n = 5). Experiments were performed as follows: from each previtellogenic female, one of the two ovaries was incubated in the medium containing a NCC extract (treated ovary), the contralateral ovary being incubated in pure medium (control ovary). Then, ecdysteroids secreted into the incubation medium by each ovary were measured using enzyme immunoassay. Results, given in Fig. 1 show that presence of extracts corresponding to 2 NCC in Grace’s medium stimulated previtellogenic ovaries to produce significantly higher amounts of ecdysteroids than their respective contralateral ovaries. We have also verified that blowfly ovaries, extracted either before or after in vitro incubations, contained no detectable amount of immunoreactive compounds (<10 pg E-eq./ovary, n = 5 for both cases). Thus, we can assume that ecdysteroids secreted in the culture media after stimulation by NCC extracts were the result of active biosynthesis. Results given in Fig. 2, expressed as the means of individual treated/control ratios, show that ecdysteroidogenic activity varied approximately as a linear function of NCC concentration, within the range tested. Because such ratios are more accurate to compare from an experiment to another and as they also minimize variations between individuals, they will be preferred in the following experiments.
8 means of treated /control r atios
294
7 6 5 4 3 2 1 0 0
1
2 3 NCC equivalents
Fig. 2. Ecdysteroidogenic activity in vitro expressed as means (±SEM, n = 4–10 per point) of individual treated/control ratios in function of NCC equivalents. Points over 1 NCC equiv. were significantly different from their respective controls. Linear regression indicated that activity could be estimated as: y = 2.1 NCC + 1.0 (r2 = 0.98).
3.2. Effect of authenticated peptides on ovarian steroidogenesis Experiments using the same in vitro test were then performed with authenticated locust neuropeptides, namely NPs, obtained from Dr. J. Girardie in two highly purified forms, NP-A and NP-B, and OMP, available in synthetic form. Results, summarized in Table 1, showed that none of these peptides had a significant steroidogenic effect on blowfly ovaries at similar concentrations than those previously tested for crude extracts. In the experiments shown in Table 1, for better comparison with our other experiments, OMP concentrations were expressed in NCC equivalents, using the approximate content of OMP in NCC measured by Dr. J Girardie (personal communication). However, as this peptide was available in synthetic form, its activity has also been tested in molar concentrations ranging from 10 9 M to 10 5 M (by a step 10): no steroidogenic activity was observed during these experiments, apart from a slight but significant activity at the highest concentration (stimulation ratio of 1.9 ± 0.3, n = 10, at 10 5 M, corresponding approximately to 25 NCC equiv. per culture), which, however, was not comparable to the high activity of NCC extracts.
200
pg ecdysone equivalents
3.3. Partial purification of NCC extracts Partial purification of NCC extracts was then undertaken in order to better identify the steroidogenic factor(s) responsible for the stimulation of blowfly ovaries. A simple analysis of extraction procedures was performed using three different solvent conditions,
100
0
ovaries before culture
0 NCC (controls)
2 NCC (treated)
ovaries after culture
Fig. 1. In vitro effects of crude NCC extracts on ovarian steroidogenesis by blowfly isolated ovaries. Ecdysteroids expressed as pg of ecdysone equivalents per ovary or per culture were measured (i) in the ovaries (n = 5) either before or after culture (‘‘before” being done on similar ovaries than those really incubated) and (ii) in the culture media after overnight incubation of ovaries (n = 10) in the absence (controls, 0 NCC) or in the presence (2 NCC equivalents) of crude NCC extracts. Mean secretion (±SEM, error bars) of ecdysteroids after stimulation by 2 NCC was significantly different from that of controls (Student t-test, P < 0.05). Values extracted in the ovaries were in both cases at the background sensitivity of the method.
Table 1 Effect of highly purified NPs (NP-A and NP-B) and of synthetic OMP on ovarian steroidogenesis in blowflies. In the case of OMP, 1 NCC-equivalent is roughly supposed to correspond to 2 ng/ll for a total volume of 50 ll incubation medium and represents ca. 4.10 7 M (personal communication from Dr. J. Girardie). None of the ratios was significantly different from the theoretical value of 1 (statistics using Student’s t-test, P < 0.05). Neuropeptides
Neuropeptide amounts in NCC equivalents
n
Results expressed as treated/control ratios
Purified NP-A Purified NP-A Purified NP-B Purified NP-B Synthetic OMP Synthetic OMP
2 5 2 5 2 (4 ng/ll) 5 (10 ng/ll)
11 7 10 10 9 20
1.2 ± 0.2 1.1 ± 0.2 1.1 ± 0.2 1.4 ± 0.2 1.3 ± 0.2 1.4 ± 0.2
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G. Manière et al. / General and Comparative Endocrinology 163 (2009) 292–297 Table 2 Steroidogenic activity of locust NCC extracts (2 NCC-equiv. per culture), after successive extractions using various solvent conditions. (): significantly different from the theoretical value of 1 (Student’s t-test, P < 0.05).
applied successively to the same NCC pools. Results presented in Table 2 show that, though some steroidogenic activity was partly soluble in 100% and 70% methanol, the main part of this activity was only soluble after an acidic ethanol extraction. Extraction in pure methanol, known to be favorable to purification of NPs and OMP (Girardie et al., 1989; 1991; 1998b), did not allowed a sufficient recovery of ecdysteroidogenic activity, confirming our above results with authenticated peptides. 3.4. HPLC of NCC peptides
NPs 0.5
OMP
1.7 ± 0.1 () 2.8 ± 0.3 () 4.4 ± 0.9 ()
0 1
B
NPs OMP
Treated/control ratios
8 8 8
absorbance
n
100% methanol 70% methanol Acidic ethanol
A
absorbance
Extraction conditions
1
0.5
CP
LIRP
3.5. Steroidogenic activity of HPLC fractions Analysis of the HPLC fractions obtained from acidic ethanol extracts, in terms of their capacity to modify ovarian ecdysteroidogenesis in our in vitro test, revealed that a high steroidogenic activity was found in a single group of fractions co-migrating with the UV-peak tentatively identified as LIRP and reached a maximum at fraction #21 that showed anti-insulin immunoreactivity (Fig. 3C). No significant activity, neither steroidogenic nor steroidostatic (i.e., inhibitory), was found in the other fractions, in particular those corresponding to NPs and OMP, confirming the observations made with authenticated peptides at the same concentrations. Incidentally, the peak corresponding to the putative C-peptide was not significantly active. Importantly, we also observed the same pattern of activity after methanol extraction
0 steroidogenic activity
Analysis of NCC extracts was then made by HPLC, using a reverse-phase procedure adapted from that described by Hétru et al. (1991), in order to facilitate the identification of the main peaks displayed by UV detector. Interestingly, UV-peaks corresponding to OMP and NPs (identified by comparison with the retention time of reference compounds) were observed whatever the extraction procedure: indeed, these neurohormones were satisfactorily recovered after methanol extraction (Fig. 3A), as well as after acidic ethanol extraction (Fig. 3B). In this second case however, OMP and NPs were accompanied by other UV-peaks, in particular by a peak which, as judged from its relative retention time, behave chromatographically very similarly as LIRP, previously identified by Hétru et al. (1991). Indeed, this peak migrated before OMP and NPs, at ca. 80% of NP retention time in similar HPLC conditions. In addition, the abundance of this peak in acidic conditions, which was the major component of NCC after NPs, and its absence from the chromatogram obtained with methanolic extracts, were in agreement with the observations of Hétru et al. (1991) and corroborated its identification as LIRP. Moreover, the HPLC fraction #21 containing most of this peak showed a low but significant affinity for anti-insulin polyclonal antibodies, confirming the presence of an insulin-like peptide in this fraction. Incidentally, acidic extraction also enabled the observation of smaller peaks, migrating before putative LIRP, possibly corresponding to truncated LIRP forms observed by Hétru et al. (1991), and of a peak with a higher retention time than NPs, probably corresponding to the C-peptide (the peptide resulting from the cleavage of A and B chains from insulin precursor), also identified by Hétru et al. (1991).
3
C 2
*
1 0
20
40
time (min) 60
Fig. 3. HPLC separation of NCC neuropeptides after (A) methanol extraction or (B) acidic ethanol extraction. Ordinate indicates absorbance at 225 nm. Abscissa indicates time in min. Retention times of OMP and NPs were confirmed by similar HPLCs made with authenticated peptides. Peaks supposed to correspond to LIRP or C-peptide (CP) were tentatively identified on the basis of the data of Hétru et al. (1991). (C) Steroidogenic activity of the fractions (1 fraction per min) corresponding to 2 NCC equivalents, separated after acidic ethanol extraction. Results (ordinate: steroidogenic activity) are expressed as treated/control ratios. As no steroidostatic activity was found, ordinate below 1 is useless. Fractions 1–17 and 31–60 are not shown (they have not been assayed one by one, but by pools that were found inactive). Errors bars indicate means ± SEM obtained from 5 to 6 EIA measurements. Points over 1.5 were significantly different from their respective controls. Asterisk indicates a fraction with slight anti-insulin immunoreactivity.
(not shown), but at a much lower level, suggesting that the lower activity obtained after this extraction protocol was only due to a lower recovery of the same active factor. A few other experiments (not shown) were also done by pooling different HPLC fractions, in particular those corresponding to LIRP plus OMP and NPs, which did not reveal supplementary activity, suggesting that possible interactions between these different peptides were not beneficial for steroidogenesis in isolated blowfly ovaries. Altogether, HPLC results confirmed that neither OMP nor NPs were able to explain the high steroidogenic activity of NCC extracts in blowflies, but demonstrated that another (and probably single) factor, migrating like LIRP, was responsible for this effect. 4. Discussion 4.1. Activity of locust NCC crude extracts on blowfly ovaries Our results first show that extracts from locust NCC are able to trigger ovarian secretion of ecdysteroids in an insect, the blowfly,
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belonging to a very distant group. This secretion is obviously due to an increase in steroidogenesis, as blowfly previtellogenic ovaries do not store significant amounts of immunoreactive ecdysteroids. The precise dissection of locust large sized NCC, washed from hemolymph contamination, and the use of various extraction procedures also confirm that biological activity was likely associated with neuropeptides. Indeed, our results might not be attributed to the artefactual presence of some ecdysteroids in NCC extracts, as indicated by the absence of immunoreactive ecdysteroids in crude extracts. More importantly, even the transformation of non-immunoreactive ecdysteroids, such as putative precursors or conjugates, into immunoreactive ones can be ruled out in our extracts. Indeed, during the various steps of NCC purification, the main part of steroidogenic activity remained in the pellets and was badly extracted by successive treatments with pure and with 70% methanol, each of them being yet considered very suitable for dissolving most steroid compounds. By contrast, activity was efficiently extracted from such pellets by acidic ethanol, a treatment that is suitable for many peptides, but that is not particularly recommended for steroids. Our HPLC results also indicate that only one group of fractions was active on blowfly ovaries. This observation is relatively surprising, as our previous studies have shown that ecdysteroidogenesis in blowfly ovaries is modulated by at least two different stimulatory signaling pathways (Manière et al., 2000), as well as by two different inhibitory ones (Manière et al., 2002, 2003). It can thus be assumed that only one of the blowfly factors has a homologue in locust NCC and that the others either have diverged too much during evolution or are produced by other site(s) of secretion. 4.2. Locust NPs and OMP are not active on blowfly ovaries Our data also indicate that NPs (either purified NP-A and NP-B prepared by Dr. J. Girardie or our own HPLC fractions co-migrating as NPs) have no significant ecdysteroidogenic effect on blowfly ovaries. Though this finding appears consistent with the antigonadotropic role of NPs previously described in locusts (Girardie et al., 1987), it is yet surprising because NPs also have some sequence similarities with OEH, the steroidogenic hormone of female mosquitoes (Brown et al., 1998). It is thus possible that NPs, OEH and their putative homologues in flies have diverged too much during evolution to bind with the same ligands or with the same receptors and to fulfill the same functions. It is also possible that NPs, which also display similarities with vertebrate insulin-like growth factor binding proteins (Badisco et al., 2007; Claeys et al., 2003) and interact in vitro with insulin-like peptides (Badisco et al., 2008), are unable to exert their action alone in blowflies, but the lack of a significant effect when NPs were pooled with LIRP containing fractions in our conditions remains difficult to interpret. However, another possibility could be that NP/OEH homologues are absent in flies and have no physiological role, as suggested by the observation that no corresponding gene has been identified so far in Drosophila genome (Hewes and Taghert, 2001; Nässel, 2002), although similar sequences have been found in various other invertebrates (Claeys et al., 2003). Our results also showed that OMP is not the locust NCC factor able to trigger acute ecdysteroidogenesis in blowfly ovaries. Indeed, though OMP seemed to have a slight ecdysteroidogenic effect at a very high concentration in our in vitro tests on blowfly ovaries, it was inactive at the concentrations at which crude extracts were already very efficient. However, if a peptide homologue of OMP would exist in blowflies, it seems highly probable that it should be active at lower concentrations. OMP has been described as a putative gonadotropin in Locusta and in Schistocerca (Girardie and Girardie, 1996; Girardie et al., 1998b) on the basis of several
in vivo evidences, but its capacity to stimulate ovarian steroidogenesis has not yet been confirmed in vitro, i.e., directly on locust ovaries. Moreover, the presence of OMP seems to be restricted to Acridae (Richard et al., 1994) and homologues or orthologues have not been described in other insects so far. Thus, although our present data do not discard the possibility for OMP to be involved in the control of ovarian ecdysteroidogenesis in locusts, they clearly show that this neuropeptide, either alone or pooled with other NCC fractions, is not responsible for the steroidogenic effect of locust NCC extracts on blowfly ovaries. 4.3. Gonadotropic role of insulin-like peptides The main positive data obtained during this study indicate that the steroidogenic factor active on blowfly ovaries has several properties suggesting it may be LIRP, the only insulin-like protein present in locust NCC according to Hétru et al. (1991). Indeed, the steroidogenic activity is mainly recovered after extraction in acidic conditions, but not after neutral ones, as it is generally the case for insulin-like proteins (Teller and Pilc, 1985), including LIRP (Hétru et al., 1991). Moreover, the only group of HPLC fractions having a steroidogenic activity clearly migrated like a very high UV peak, corresponding, in terms of its relative abundance and retention time, to a compound previously identified as LIRP by Hétru et al. (1991) in a similar separation system. Lastly, anti-insulin-like immunoreactivity was found in the HPLC fraction having the highest steroidogenic activity. Since NCC contain only three major neurohormones and since the steroidogenic factor is neither OMP nor NPs, then the possibility that it could be the third one, i.e., LIRP, appears very likely. By contrast, the possibility that the steroidogenic factor could be a minor compound migrating in the same fractions as LIRP, although not definitively ruled out by our experiments, appears less credible. Unfortunately, we have been compelled to interrupt this work on insect ovarian steroidogenesis before the complete identification of the active factor. Further studies in other laboratories will be necessary to obtain a definitive proof, requiring the complete purification or the chemical synthesis of LIRP and the verification of its activity in similar bioassays. Nevertheless, our present experimental data are strongly in agreement with our previous studies having already shown that exogenous insulin-like peptides from distant animals, namely bovine insulin and bombyxin II, as well as endogenous insulin-like peptides from medial neurosecretory cells, are able to stimulate ecdysteroidogenesis in blowfly isolated ovaries (Manière et al., 2004). Moreover, they are also in good agreement with several observations in some other insects suggesting that insulin-like peptides have a steroidogenic role during oogenesis (reviewed in Claeys et al., 2002; Garofalo, 2002; Brown et al., 2009), as well as during post-embryonic development. In conclusion, a peptide from locust NCC has a steroidogenic activity on blowfly ovaries. Though this factor has not been definitely identified, the present results show that it is neither NPs nor OMP, but probably LIRP, in agreement with our previous observations showing that insulin-like peptides are major steroidogenic gonadotropins in blowflies. Further studies are needed to verify whether this property of insulin-like peptides, only observed in mosquitoes and flies (review in Brown et al., 2009), can be generalized to other insect groups and, more particularly, whether LIRP also has such a function in locusts. Acknowledgments The authors thank Josiane and Adrien Girardie (now retired) for their help and support during this study. They also thank M. De Reggi (Marseille), for the gift of anti-ecdysone antibodies.
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