The NMDA receptor antagonist MK-801 inhibits vitellogenesis in the flesh fly Neobellieria bullata and in the desert locust Schistocerca gregaria

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Abstract

We found that in the flesh fly Neobellieria bullata, vitellogenesis can be inhibited in a dose-dependent way by two injections of 60 μg MK-801/g body mass. In the desert locust Schistocerca gregaria, vitellogenesis can also be fully inhibited but only by repeated injections of 200–400 μg/g body mass. In this species, the inhibition can be overruled by coapplication of juvenile hormone. Vitellogenin bands remained visible in electropherograms of hemolymph of MK-801-treated female locusts, but vitellogenin did not accumulate as might be expected when only its uptake by the oocytes, and not its synthesis by the fat body, would be affected. Whether MK-801 acts by inhibiting juvenile hormone synthesis by the corpora allata remains to be investigated.

Introduction

For a long time, juvenile hormone (JH) was thought to be the gonadotropic hormone of insects. This was concluded from the fact that in numerous species in which vitellogenin accumulation in the oocytes starts in the adult stage, allatectomy inhibits ovarian development and that application of synthetic JH overrules the effect of this ablation. To date, we know that the situation is more complex. In addition to JH, gonadotropins produced by neurosecretory cells in the brain belonging to the parsin-, insulin-, and short NPF-families of neuropeptides play a role, as does 20-OH ecdysone (De Loof and Huybrechts, 1999; De Loof et al., 2001; Claeys et al., 2002). The search for brain factors that may be involved in controlling JH biosynthesis by the corpora allata resulted in the discovery of numerous allatostatins and allatotropins (Stay, 2000; Tobe et al., 2000). Their effect on JH biosynthesis is often species-specific. In addition to neuropeptides, some classical neurotransmitters may also play a role, perhaps with an indirect effect on reproduction as a result. Recently, some evidence for novel functions controlled by glutamate was reported in both vertebrates and insects, not by direct application of glutamate, but by interfering with a glutamate receptor.

Repeated injections of MK-801, an antagonist of the N-methyl-D-aspartate glutamate receptor (NMDAR) retards puberty in female, but not in male platyfish Xiphophorus maculatus. These and other data made Flynn et al. (2002) conclude that in this fish model the NMDAR influences reproductive development at, or above the level of gonadotropin releasing hormone (GnRH)-containing cells of the brain–pituitary–gonadal axis. In insects, the search for peptidic counterparts of vertebrate GnRHs did not yet yield conclusive results. A radioimmunological search using antibodies against different GnRHs did not reveal positive signals in brain extracts of locusts (De Loof et al., 1995), indicating absence of a substantial degree of homology. Neither did database searches using conventional programs uncover GnRH-like peptides in Drosophila or Anopheles. With respect to GnRH receptors, Hauser et al. (1998) found that Drosophila melanogaster has a receptor that is structurally related to GnRH receptors. Remarkably, not an GnRH-like peptide but adipokinetic hormone (AKH) was found to be its endogenous ligand (Staubli et al., 2002).

Again in D. melanogaster, the NMDAR has been immunolocalized by Chiang et al. (2002). Using mouse monoclonal antibody against rat NMDAR1 protein, they visualized two neurons in the lateral protocerebrum, one per hemisphere, with extensions ending in extensive arborizations toward the pars intercerebralis (PI). The PI of insects is thought to be the site of synthesis of gonadotropins. There was also labeling in one of the three types of endocrine cells of the ring gland, namely the CA cells. By means of reverse transcription PCR, two mRNA transcripts, DNMDAR1and DNMDAR2, could be demonstrated in the ring gland and brain of last-instar D. melanogaster. Chiang et al. (2002) also tested the effect of MK-801 on JH production by isolated corpora allata of the cockroach Diploptera punctata. MK-801 significantly reduced the stimulatory effect of NMDA on JH biosynthesis, adding strength to the view that NMDA receptors are present in the corpora allata and actively contribute to control JH biosynthesis.

Before engaging in labor-intensive molecular studies on the possible involvement of the NMDAR in control of reproduction in insects, it should be investigated whether, like in platyfish, injections of MK-801 inhibit ovarian development. We here describe the results of such experiments in the flesh fly Neobellieria bullata and in the desert locust Schistocerca gregaria as well as those of a rescuing experiment with JH.

Section snippets

Chemicals

The NMDAR antagonist (5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801), as well as JH III, were purchased from Sigma, USA.

Experimental animals

The grey flesh fly N. bullata (formerly called Sarcophaga bullata) was reared as described by Huybrechts and De Loof (1982). Four–day old female flies that had been fed on water and sugar were used in the experiments. The desert locust S. gregaria was reared under crowded conditions according to Rahman et al. (2002). For the

Effect of MK-801 in N. bullata

Significant differences in ovarian development were observed between MK-801-treated and control animals (Fig. 1). In the control, most (60%) of the females showed ovaries with oocytes occupying 50% of the length of the follicles (stage 4B). Injection of a dose of 5 μg MK-801/gbm caused a significant decrease in the percentage of females being in stage 4B. Upon increasing the dose to 20 and 40 μg MK-801/gbm, the ovaries of most of the females did not develop beyond the previtellogenic stage. The

Discussion

The NMDAR contributes to the regulation of reproduction in rats (Urbanski and Ojeda, 1990; Gore et al., 1996) and platyfish (Flynn et al., 2002, Flynn et al., 1999). Our results demonstrate that the same holds true for some insect species as well because injection of the antagonist MK-801 clearly inhibits, in a dose-dependent manner, ovarian development in both the heterometabolous S. gregaria and the holometabolous N. bullata. This raises the question whether, perhaps, there is a common

Acknowledgements

The authors acknowledge funding by a FWO-Grant (G.0143.03) and a GOA-Grant (GOA/2000/04) from the research council of the K.U. Leuven.

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