- Email: [email protected]
Schistosome male–female interaction: induction of germ-cell differentiation
Review
TRENDS in Parasitology Vol.17 No.5 May 2001
227
Schistosome male–female interaction: induction of germ-cell differentiation Werner Kunz Male and female schistosomes are permanently paired while they live in the bloodstream of their vertebrate hosts. Female schistosomes produce eggs only when they are in intimate association with a male. Here, I combine classical cytological knowledge about the cellular processes in the female that are affected by the male with recent molecular results that are beginning to allow speculation about the signalling events involved.
Schistosomes have a unique biological feature: they reside as permanently paired couples in the bloodstream of humans, often for many years. This intimate contact is a prerequisite for female reproductive development and for the maintenance of her mature state1. Change of mate can occur, although mating is usually monogamous2. Surprisingly, after the pioneering work of Paul Basch and Irene Popiel in the 1980s1, this important finding has been largely neglected by researchers3. Such permanent associations are very unusual in the animal kingdom. A comparable phenomenon is found in Diplozoon (monogenean trematodes), in which the two hermaphrodite partners are not only paired but also become fused at the point of contact, and their genital ducts are permanently joined. Another example is Bonellia viridis, a higher worm (Echiurida), whose isolated larvae almost all develop into females. If, however, a larva comes into contact with the proboscis of an adult female, it becomes male, developing testes and starting spermatogenesis within just a few days. Differentiation into males can also be experimentally induced in isolated larvae by incubation with extracts from the female proboscis4. Almost nothing is known about the molecular mechanisms regulating these developmental induction processes. In 1990, Basch wrote in Parasitology Today: ‘I consider it likely that there actually is sexual interdependence, possibly with reciprocal chemical messengers, between male and female schistosomes. However, after 70 years or so of searching, nobody has satisfactorily demonstrated that such a substance exists and nothing at all is known about any putative receptor or possible mechanism’5. Werner Kunz Genetic Parasitology Group, Institute of Genetics, Heinrich-Heine University, Universitätsstr. 1, D-40225 Düsseldorf, Germany. e-mail: [email protected]
Targets of male stimulation
In female schistosomes, an intimate and permanent association with the male is necessary for reproduction to occur. Virgin females that grow up in the absence of males differ considerably in size from those recovered from mixed infections in which they were paired with males (Fig. 1a,b) and do not develop eggs. Similarly, when egg-laying female schistosomes
are separated from their male partners and surgically reimplanted into the host, they stop laying eggs and begin to regress reproductively to the immature state. However, if such regressed females are allowed to pair again with males, normal reproductive activity resumes even after months of regression. By contrast, reproductive development in the male is not significantly influenced by the presence of the female6. The design of molecular strategies to study male stimulation of females requires an understanding of the histology and cytology of female reproductive development. In schistosomes, as in other platyhelminths, the female gonad is divided into an ovary and a so-called vitelline gland. The ovary produces oocytes that are eventually fertilized and are destined to continue the germ line. The vitelline gland produces additional cells that are joined with the oocyte into a compound egg but that are degenerated during the early cleavage divisions and do not take part in embryonic development. The name vitelline gland, however, is misleading because this organ produces only small amounts of vitelline substances7; it predominantly synthesizes eggshell proteins8. Ontogenically, it is assumed that both oocytes and vitellocytes arise from common mother cells. Consequently, vitellocytes can be considered as abortive germ cells, analogous to the nurse cells in the ovaries of higher insects. Ultrastructural work by Erasmus in the 1970s and early 1980s provided insight into the cellular processes in the female that are influenced by the male. Developmental repression in virgin females from unisexual infections mainly concerns the ovary and the vitelline gland; other parts of the female reproductive system (e.g. uterus, oviduct and vitelline duct) and the ootype and Mehlis’ glands develop normally6. This observation has important implications: it shows that the male factor affects very specific cellular events in the female. The ovary and the vitelline gland are very small in virgin females and barely visible, although they are clearly present. The male signal therefore does not differentiate pluripotent stem cells into germ cells. Instead, it regulates the multiplication and differentiation of germ cells that are already present, located in separate organs and committed to become oocytes or vitellocytes before pairing with a male. Almost no detailed information is available on the cytological events that are initiated in the ovary by the male signal and so the following observations and
http://parasites.trends.com 1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)01893-1
Review
228
TRENDS in Parasitology Vol.17 No.5 May 2001
Females of S. mansoni
(b) Paired with mansoni
(c) Paired with japonicum Ovary
(d) Paired with haematobium Ovary
(a) Virgin
Ovary
Vitelline gland
Vitelline gland
From these cytological observations, it can be hypothesized that at least two signalling pathways are involved: one that induces S phase and increases the number of Stage-1 vitellocytes; and a second that initiates unequal, differential mitotic cell divisions that result in daughter cells that enter the final pathway of terminal cell differentiation (Fig. 2). The differentiation into Stage 2, Stage 3 and finally Stage 4 vitellocytes is visible cytologically9. At the molecular level, it has been shown that a number of genes expressed in these differentiating vitellocytes are strictly controlled in response to worm pairing12,13. Interruption of pairing stops transcription, whereas re-mating restores their expression. This contrasts with other schistosome genes, which are expressed in both genders and are not controlled by pairing with the male12. Nature of the male stimulus
Gut
TRENDS in Parasitology
Fig. 1. Virgin females of Schistosoma mansoni that grew up in the absence of male worms (a) remain immature and small, and do not lay eggs. They differ considerably in size and body shape from females that were paired with S. mansoni males (b). S. mansoni females paired with males of a different species (c,d) also grow up to adulthood and fertility but, in most such cases, they remain distinctly smaller than females from homospecific matings. The dark structures visible in virgin females mostly represent the gut, whereas, in paired females, the vitellarium is also clearly visible. Modified, with permission, from Ref. 36. Scale bar = 1 mm.
conclusions concern only vitellocyte development, although oocytes might be similarly regulated. The vitellaria of virgin females are undifferentiated and contain low numbers of vitellocytes. According to Erasmus’ categorization9, all vitellocytes in unisexual or separated females are Stage 1 cells – that is, undifferentiated stem cells10. These cells are the target of the male stimulation. Pairing with a male induces a considerable increase in the number of vitellocytes as well as the final initiation of terminal cell differentiation of a selected number of vitellocytes, which develop into Stage 2, Stage 3 and finally Stage 4 cells9 (Fig. 2). It has been shown autoradiographically that paired females incorporate much more [3H]-thymidine than females from single-sex infections11. The most densely labelled nuclei were Stage 1 vitellocytes. If mature egglaying females were separated from their paired males, these females had a continual decrease in uptake of [3H]-thymidine compared with females maintained with male partners. This uptake increased again when isolated females were re-mated with males. The results show that the male worm has an effect on DNA synthesis in the vitellocytes of the female and, in addition, that a mitogenic signalling pathway is switched on in the vitellocytes when the female receives the developmental stimulus from the male. http://parasites.trends.com
The intimate and permanent association between male and female worms necessary for reproduction to occur is known to be independent of sperm transfer14. In an attempt to localize the stimulatory factor, male worms were cut into segments and incubated with virgin females that paired in vitro with the male segments15. Vitelline gland development occurred only in that small segment of the female that was in direct physical contact with the male segment. Furthermore, only the portions in contact with cut male segments showed significantly higher uptake of [3H]-thymidine (indicating increased DNA synthesis and cell division) than the non-contacted portions. This shows that each part of the male body along the anterior–posterior axis can stimulate the female and that the female developmental response is not propagated longitudinally. If Schistosoma mansoni females are paired with male partners of a different species, several such heterospecific pairings result in females that, although fertile, produce reduced numbers of eggs16. In addition, they remain smaller than females from homospecific pairs (Fig. 1c,d). Thus, the male stimulus acts to a certain extent in a species-specific manner. A purely tactile stimulus seems to be inconsistent with these observations because it would not be expected to be species specific. It is more likely that the stimulation involves a chemical transfer. Acetone or ether extracts from males could initiate the development of vitellocytes in vitro in females from single-sex infections. It has thus been suggested that lipids might be involved in the transfer of the male signal17. Male extracts appear to enhance the transcription of the major eggshell-coding gene p14 in virgin females18. The expression of this gene under normal conditions occurs in females only when they are paired12. Cholesterol has been shown to be transferred between male and female adult schistosomes19. From these observations, it has been speculated that steroid hormones might be important
Review
Fig. 2. Hypothetical scheme of the differentiation processes in the vitellarium that are induced by pairing with the male. The vitellaria of virgin females contain low numbers of vitellocytes, and these are all undifferentiated Stage 1 cells9. These cells are the target of the male stimulation. From the morphological data, at least two signalling pathways are probably involved: one that induces S phases and a multiplication in number of Stage-1 vitellocytes; and a second that initiates unequal mitotic cell divisions, resulting in daughter cells that differentiate into Stage 2, Stage 3 and finally Stage 4 cells.
TRENDS in Parasitology Vol.17 No.5 May 2001
Male signal
Stage 1
Equal cell division
Mitogenic genes expressed
Stage 1
Unequal cell division
Stage 1
Stage 2 Stage 1
Genes for differentiation expressed Terminal differentiation Stage 3
Stage 4 TRENDS in Parasitology
in male–female interactions20. Recently, a steroidhormone receptor has been identified that binds to promoter elements of the p14 gene21. Previous reports have described the occurrence of ecdysones in adult schistosomes22. However, the peaks of synthetic activity of ecdysone in schistosome development militate against its having role in male–female interaction: the highest concentrations of free ecdysteroid were found in lung-stage worms, not in adults22. Ecdysteroids were originally characterized as the moulting hormones of insects and, following recent taxonomy, are characteristic for a division of protostomes called the Ecdysozoa, which includes nematodes and arthropods. Only these groups exhibit ecdysone-regulated moulting, whereas the metabolic function of ecdysteroid in the other large protostome clade, the Lophotrochozoa (which includes the platyhelminths), is still not clear23. This would call into question a regulatory role for ecdysone in schistosome development; as a consequence, there should be no nuclear ecdysone receptor regulating the effects of ecdysteroids in schistosomes, because this would be considered to be a characteristic marker of the Ecdysozoa24. It might be important for an understanding of the nature of the male stimulus that schistosome females from single-sex infections occasionally develop some differentiated vitelline cells and even some malformed eggs with shells, despite the complete absence of males25. This, however, requires very long incubation times of unisexual females in the final host, and there is considerable variation between populations recovered from different mouse strains. http://parasites.trends.com
229
Nevertheless, these observations demonstrate a limited autonomous ability of vitellocytes to differentiate, although the development of a normal number of vitellocytes requires the permanent stimulus given by the male. There is still another observation with potential significance for understanding the nature of developmental induction stimulated by the male. Homosexual pairings of mature males can induce vitellocyte development up to Stage 4 in virgin males when the virgin male is located in the gynecophoric channel of the adult male partner for long enough26. The vitellocytes appear at the same position as in the female. If one considers this unusual abnormality in a phylogenetic context, it is tempting to interpret this as an evolutionary atavism. Schistosomes evolved from monoecious ancestors and, in most monoecious Digenea, the male reproductive system matures before the female system (protandry), which helps to avoid self-fertilization. In such proterandrous organisms, maturation of the female reproductive system is developmentally repressed as long as the male system is functionally active. Later in development, a de-repression mechanism specifically stimulates the differentiation of female reproductive cells. It is possible that, in recent evolutionary time, the male schistosome as a separate sex has taken over the role of de-repressing developmental events that, in the hermaphroditic ancestors, occurred in late development. The nature of male stimulation in schistosomes differs distinctly from the well-known mechanism of male-controlled egg maturation in Drosophila females27. In this species, a sex peptide is synthesized by the male and transferred to the female during copulation. If sexually mature females do not copulate with males, the production of new eggs is prevented by controlled resorption (apoptosis) of oocytes. The sex peptide in Drosophila controls vitellogenic oocyte maturation at a final stage of oogenesis during the passage of oocytes from Stage 9 to Stage 10, shortly before oviposition. By contrast, in schistosomes, the male signal molecule controls earlier events in vitellocyte differentiation. Signal transduction pathways in the female
How can a chemical signal from the male induce cellular processes in the vitelline gland inside the female body? As a first step, the signal has to cross the surface of the female. Schistosomes are not surrounded by a cuticula but are instead covered by a tegument, which is a cytologically unusual structure. The tegument is a syncytium bounded externally by two lipid bilayer membranes28. Our knowledge of the biochemistry of the schistosome surface is still in its infancy. Rapid internalization of fluorescent lipids and their analogues throughout the membrane has been seen (particularly of the lipid ceramide, which mediates cell signalling in various eukaryotes), suggesting that there is a transport mechanism
230
Acknowledgements I thank Christoph Grevelding and Khalid Zemzoumi (both at the University of Düsseldorf, Germany) for valuable ideas and suggestions. Dedicated to Paul Basch in appreciation of his essential contribution to understanding male–female interactions in schistosomes.
Review
TRENDS in Parasitology Vol.17 No.5 May 2001
through the membrane29. This transport has been observed to be restricted to distinct patches at the surface, showing that the surface of schistosomes is not homogeneous but has specialized microdomains that allow non-polar lipids to pass through. Interestingly, other studies30 have shown that the schistosome surface membrane contains caveolaelike structures – flask-like membrane microdomains that maintain a protein concentration different from that in surrounding membrane. In mammals and other eukaryotes, caveolae are suggested to be centres through which signal-transducing events are focused via a linker protein called caveolin31. In schistosomes, caveolae-like domains are mainly found in the tubercles, which are specialized areas on the dorsal surface of the male. Interestingly, an S. mansoni receptor protein serine/threonine kinase of the transforming growth factor receptor family has been colocalized to the tubercles on the dorsal surface of male worms32. It is thus tempting to speculate that, in schistosomes, as in other organisms, caveolae might also be focal points for receptors involved in signal transduction. However, the serine/threonine kinase receptor in schistosomes is detected only in adult males and is probably not directly related to the male–female interaction processes. Transport of a male signal from the tegument to the vitelline gland would be impeded by the fact that schistosomes, like other platyhelminths (being Parenchymia), are completely filled with parenchyma, lacking any kind of body cavity or circulatory fluid suited for molecular transport. The question thus arises of whether the signal is transported through intercellular spaces or whether it moves across membranes. In Drosophila, the Gurken protein, which is a growth factor that acts as a ligand to a receptor tyrosine kinase, is released from the oocyte and internalized by the follicle cells during
References 1 Popiel, I. and Basch, P.F. (1984) Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments. J. Exp. Zool. 232, 141–150 2 Tchuem Tchuenté, L.A. et al. (1996) Mating behaviour in schistosomes: are paired worms always faithful? Parasitol. Today 12, 231–236 3 Ribeiro-Paes, J.T. and Rodrigues, V. (1997) Sex determination and female reproductive development in the genus Schistosoma: a review. Rev. Inst. Med. Trop. São Paulo 39, 337–344 4 Leutert, R. (1974) Sex differentiation and gametogenesis in Bonellia viridis Rolando. J. Embryol. Exp. Morphol. 32, 169–193 5 Basch, P.F. (1990) Single sex schistosomes and chemical messengers. Reply. Parasitol. Today 6, 298 6 Erasmus, D.A. (1973) A comparative study of the reproductive system of mature, immature and ‘unisexual’ female Schistosoma mansoni. Parasitology 67, 165–183 7 Schüßler, P. et al. (1995) An isoform of ferritin as a component of protein yolk platelets in Schistosoma mansoni. Mol. Reprod. Dev. 41, 325–330 http://parasites.trends.com
signalling33. This is, however, only a bilateral cell–cell interaction of adjacent cells and not a signal transport mechanism that connects different organs with each other. It is noteworthy, however, that most schistosome tissues (and not just the tegument) are syncytial in nature. Syncytial organization might be essential for the movement of signal as well as nutritive molecules34 in organisms that depend on diffusion as a mechanism for substrate transport. Conclusions and future directions
Owing to their importance in cancer research, there are many drugs available that more-or-less specifically inhibit signal-transduction molecules. This opens a way to relate cellular target processes in schistosome reproductive development, such as vitellocyte multiplication or differentiation, with some signalling molecules that might play a role in regulating these processes. cDNA libraries can be screened for the mRNA sequences of these signalling molecules and the clones obtained used for RNA interference to achieve a more specific inhibition than is possible with chemical blockers. When the expression of transgenes in schistosomes (which at present has been applied with some preliminary success35) becomes a reality, very specific investigations of gene function will be possible. As soon as signalling molecules that govern schistosome reproductive development are identified, their partners in the signal transduction cascade should be identified using the yeast two-hybrid system. The identification and inhibition of male-induced signalling molecules in the reproductive organs of female schistosomes might open alternative ways to control schistosomiasis, because this would inhibit egg laying and therefore stop pathogenesis in infected humans. Prevention of egg production would also interrupt the propagation of this parasite.
8 Köster, B. et al. (1988) Identification and localization of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni. Mol. Biochem. Parasitol. 31, 183–198 9 Erasmus, D.A. (1975) Schistosoma mansoni: development of the vitelline cell, its role in drug sequestration, and changes induced by astiban. Exp. Parasitol. 38, 240–256 10 Popiel, I. et al. (1984) The morphology and reproductive status of female Schistosoma mansoni following separation from male worms. Int. J. Parasitol. 14, 183–190 11 Den Hollander, J.E. and Erasmus, D.A. (1984) Schistosoma mansoni: DNA synthesis in males and females from mixed and single-sex infections. Parasitology 88, 463–476 12 Grevelding, C.G. et al. (1997) Female-specific gene expression in Schistosoma mansoni is regulated by pairing. Parasitology 115, 635–640 13 Kunz, W. et al. (1995) Schistosoma mansoni: control of female fertility by the male. Mem. Inst. Oswaldo Cruz 90, 185–189 14 Basch, P.F. and Basch, N. (1984) Intergeneric reproductive stimulation and parthenogenesis in Schistosoma mansoni. Parasitology 89, 369–376 15 Basch, P.F. (1988) Schistosoma mansoni: nucleic
16 17
18
19
20
21
acid synthesis in immature females from singlesex infections, paired in vitro with intact males and male segments. Comp. Biochem. Physiol. B 90, 389–392 Jourdane, J. et al. (1995) Parthenogenesis in Schistosomatidae. Parasitol. Today 11, 427–430 Shaw, J.R. et al. (1977) Schistosoma mansoni: in vitro stimulation of vitelline cell development by extracts of male worms. Exp. Parasitol. 42, 14–20 Rumjanek, F.D. et al. (1987) Genomic regulation in immature females of Schistosoma mansoni. Mem. Inst. Oswaldo Cruz 82 (Suppl. 4), 209–212 Haseeb, M.A. et al. (1985) The uptake, localization and transfer of [4-14C]cholesterol in Schistosoma mansoni males and females maintained in vitro. Comp. Biochem. Physiol. A 82, 421–423 Silveira, A.M.S. et al. (1986) Transfer of [14C] cholesterol and its metabolites between adult male and female worms of Schistosoma mansoni. Comp. Biochem. Physiol. B 85, 851–857 Freebern, W.J. et al. (1999) Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression. J. Biol. Chem. 274, 4577–4585
Review
22 Nirde, P. et al. (1983) Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite Schistosoma mansoni. FEBS Lett. 151, 223–227 23 Knoll, A.H. and Carroll, S.B. (1999) Early animal evolution: emerging views from comparative biology and geology. Science 284, 2129–2137 24 de Mendonca, R.L. et al. (2000) Hormones and nuclear receptors in schistosome development. Parasitol. Today 16, 233–240 25 Shaw, J.R. and Erasmus, D.A. (1981) Schistosoma mansoni: an examination of the reproductive status of females from single sex infections. Parasitology 82, 121–124 26 Armstrong, J.C. (1965) Mating behavior and development of schistosomes in the mouse. J. Parasitol. 51, 605–616 27 Soller, M. et al. (1999) Control of oocyte maturation in sexually mature Drosophila
TRENDS in Parasitology Vol.17 No.5 May 2001
females. Dev. Biol. 208, 337–351 28 Hockley, D.J. and McLaren, D.J. (1973) Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. Int. J. Parasitol. 3, 13–25 29 Redman, C.A. and Kusel, J.R. (1996) Distribution and biophysical properties of fluorescent lipids on the surface of adult Schistosoma mansoni. Parasitology 113, 137–143 30 Racoosin, E.L. et al. (1999) Caveolae-like structures in the surface membrane of Schistosoma mansoni. Mol. Biochem. Parasitol. 104, 285–297 31 Parton, R.G. (1996) Caveolae and caveolins. Curr. Opin. Cell Biol. 8, 542–548 32 Davies, S.J. et al. (1998) A divergent member of the transforming growth factor β receptor family
231
33
34
35
36
from Schistosoma mansoni is expressed on the parasite surface membrane. J. Biol. Chem. 273, 11234–11240 Peri, F. et al. (1999) Local Gurken signaling and dynamic MAPK activation during Drosophila oogenesis. Mech. Dev. 81, 75–88 Skelly, P.J. et al. (1998) Glucose transport and metabolism in mammalian-stage schistosomes. Parasitol. Today 14, 402–406 Davis, R.E. et al. (1999) Transient expression of DNA and RNA in parasitic helminths by using particle bombardment. Proc. Natl. Acad. Sci. U. S. A. 96, 8687–8692 Vogel, H. (1941) Ueber den Einfluss des Geschlechtspartners auf Wachstum und Entwicklung bei Bilharzia mansoni und bei Kreuzpaarungen zwischen verschiedenen Bilharzia-Arten. Zentralbl. Bakteriol. (Abt. I) 148, 78–96
Mass spectrometric strategies: providing structural clues for helminth glycoproteins Stuart M. Haslam, Howard R. Morris and Anne Dell Here we review current knowledge of helminth glycans and introduce parasitologists to the power of the mass spectrometric techniques that have been largely responsible for defining their carbohydrate moieties. A brief overview of glycosylation in other eukaryotes is presented, with a focus on mammalian glycosylation, to facilitate understanding of how parasite structures might be recognized as ‘self’ or ‘foreign’ by the immune system of the host.
Stuart M. Haslam Howard R. Morris Anne Dell* Dept of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK SW7 2AY. *e-mail: [email protected]
Glycoproteins are abundant constituents of the outer surfaces and the excreted–secreted antigens of helminth parasites. Their structurally complex carbohydrate chains are prime candidates for host–parasite recognition events at all stages of parasitism. Indeed, glycoconjugates are increasingly being implicated in immune responses to parasite infections. In terms of their glycobiology, probably the most studied group of parasites is the schistosomes. For example, glycans carrying the Lewisx epitope have been shown to contribute to the known T helper type 1 (Th1) cell downregulation associated with schistosomiasis in both mice1 and humans2, in the induction of host cytotoxic antibodies that lead to the lysis of host cells3–5 and to the induction of hepatic granuloma6,7. However, little is known about the molecular basis of carbohydrate-mediated host–parasite interactions. This is largely because the structures of relatively few helminth carbohydrates have been rigorously defined and even fewer are
available in quantities that permit immunological functional investigations. High-sensitivity mass spectrometry (MS) provides a means of directly addressing the first of these issues and, indirectly, the second; by amassing an increasing number of structurally defined parasite glycans we can compare structural relationships to host molecules and start to predict their potential functionality. N- and O-glycosylation
There are two main types of protein glycosylation: N-glycosylation, in which the glycan is attached to an Asn residue present in the tripeptide consensus sequon Asn-X-Ser/Thr (where X can be any amino acid except Pro), and O-glycosylation, in which the glycan is attached to a Ser or Thr residue (reviewed in Refs 8,9). Glycoproteins can contain just N- or O-glycans, or a combination of both. N-glycans are synthesized from a common precursor (Fig. 1), which is processed by stepwise trimming and stepwise addition of new sugar residues. Trimming by α-glucosidases and α-mannosidases without any subsequent glycosyl addition to the periphery results in glycans having the composition Man5-9GlcNAc2. Glycans of composition Man5-9GlcNAc2 are designated ‘high mannose’ or ‘oligomannose’. In invertebrates, it is not uncommon for additional α-mannose residues to be removed, giving glycans
http://parasites.trends.com 1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0169-4758(00)01860-3
TRENDS in Parasitology Vol.17 No.5 May 2001
227
Schistosome male–female interaction: induction of germ-cell differentiation Werner Kunz Male and female schistosomes are permanently paired while they live in the bloodstream of their vertebrate hosts. Female schistosomes produce eggs only when they are in intimate association with a male. Here, I combine classical cytological knowledge about the cellular processes in the female that are affected by the male with recent molecular results that are beginning to allow speculation about the signalling events involved.
Schistosomes have a unique biological feature: they reside as permanently paired couples in the bloodstream of humans, often for many years. This intimate contact is a prerequisite for female reproductive development and for the maintenance of her mature state1. Change of mate can occur, although mating is usually monogamous2. Surprisingly, after the pioneering work of Paul Basch and Irene Popiel in the 1980s1, this important finding has been largely neglected by researchers3. Such permanent associations are very unusual in the animal kingdom. A comparable phenomenon is found in Diplozoon (monogenean trematodes), in which the two hermaphrodite partners are not only paired but also become fused at the point of contact, and their genital ducts are permanently joined. Another example is Bonellia viridis, a higher worm (Echiurida), whose isolated larvae almost all develop into females. If, however, a larva comes into contact with the proboscis of an adult female, it becomes male, developing testes and starting spermatogenesis within just a few days. Differentiation into males can also be experimentally induced in isolated larvae by incubation with extracts from the female proboscis4. Almost nothing is known about the molecular mechanisms regulating these developmental induction processes. In 1990, Basch wrote in Parasitology Today: ‘I consider it likely that there actually is sexual interdependence, possibly with reciprocal chemical messengers, between male and female schistosomes. However, after 70 years or so of searching, nobody has satisfactorily demonstrated that such a substance exists and nothing at all is known about any putative receptor or possible mechanism’5. Werner Kunz Genetic Parasitology Group, Institute of Genetics, Heinrich-Heine University, Universitätsstr. 1, D-40225 Düsseldorf, Germany. e-mail: [email protected]
Targets of male stimulation
In female schistosomes, an intimate and permanent association with the male is necessary for reproduction to occur. Virgin females that grow up in the absence of males differ considerably in size from those recovered from mixed infections in which they were paired with males (Fig. 1a,b) and do not develop eggs. Similarly, when egg-laying female schistosomes
are separated from their male partners and surgically reimplanted into the host, they stop laying eggs and begin to regress reproductively to the immature state. However, if such regressed females are allowed to pair again with males, normal reproductive activity resumes even after months of regression. By contrast, reproductive development in the male is not significantly influenced by the presence of the female6. The design of molecular strategies to study male stimulation of females requires an understanding of the histology and cytology of female reproductive development. In schistosomes, as in other platyhelminths, the female gonad is divided into an ovary and a so-called vitelline gland. The ovary produces oocytes that are eventually fertilized and are destined to continue the germ line. The vitelline gland produces additional cells that are joined with the oocyte into a compound egg but that are degenerated during the early cleavage divisions and do not take part in embryonic development. The name vitelline gland, however, is misleading because this organ produces only small amounts of vitelline substances7; it predominantly synthesizes eggshell proteins8. Ontogenically, it is assumed that both oocytes and vitellocytes arise from common mother cells. Consequently, vitellocytes can be considered as abortive germ cells, analogous to the nurse cells in the ovaries of higher insects. Ultrastructural work by Erasmus in the 1970s and early 1980s provided insight into the cellular processes in the female that are influenced by the male. Developmental repression in virgin females from unisexual infections mainly concerns the ovary and the vitelline gland; other parts of the female reproductive system (e.g. uterus, oviduct and vitelline duct) and the ootype and Mehlis’ glands develop normally6. This observation has important implications: it shows that the male factor affects very specific cellular events in the female. The ovary and the vitelline gland are very small in virgin females and barely visible, although they are clearly present. The male signal therefore does not differentiate pluripotent stem cells into germ cells. Instead, it regulates the multiplication and differentiation of germ cells that are already present, located in separate organs and committed to become oocytes or vitellocytes before pairing with a male. Almost no detailed information is available on the cytological events that are initiated in the ovary by the male signal and so the following observations and
http://parasites.trends.com 1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)01893-1
Review
228
TRENDS in Parasitology Vol.17 No.5 May 2001
Females of S. mansoni
(b) Paired with mansoni
(c) Paired with japonicum Ovary
(d) Paired with haematobium Ovary
(a) Virgin
Ovary
Vitelline gland
Vitelline gland
From these cytological observations, it can be hypothesized that at least two signalling pathways are involved: one that induces S phase and increases the number of Stage-1 vitellocytes; and a second that initiates unequal, differential mitotic cell divisions that result in daughter cells that enter the final pathway of terminal cell differentiation (Fig. 2). The differentiation into Stage 2, Stage 3 and finally Stage 4 vitellocytes is visible cytologically9. At the molecular level, it has been shown that a number of genes expressed in these differentiating vitellocytes are strictly controlled in response to worm pairing12,13. Interruption of pairing stops transcription, whereas re-mating restores their expression. This contrasts with other schistosome genes, which are expressed in both genders and are not controlled by pairing with the male12. Nature of the male stimulus
Gut
TRENDS in Parasitology
Fig. 1. Virgin females of Schistosoma mansoni that grew up in the absence of male worms (a) remain immature and small, and do not lay eggs. They differ considerably in size and body shape from females that were paired with S. mansoni males (b). S. mansoni females paired with males of a different species (c,d) also grow up to adulthood and fertility but, in most such cases, they remain distinctly smaller than females from homospecific matings. The dark structures visible in virgin females mostly represent the gut, whereas, in paired females, the vitellarium is also clearly visible. Modified, with permission, from Ref. 36. Scale bar = 1 mm.
conclusions concern only vitellocyte development, although oocytes might be similarly regulated. The vitellaria of virgin females are undifferentiated and contain low numbers of vitellocytes. According to Erasmus’ categorization9, all vitellocytes in unisexual or separated females are Stage 1 cells – that is, undifferentiated stem cells10. These cells are the target of the male stimulation. Pairing with a male induces a considerable increase in the number of vitellocytes as well as the final initiation of terminal cell differentiation of a selected number of vitellocytes, which develop into Stage 2, Stage 3 and finally Stage 4 cells9 (Fig. 2). It has been shown autoradiographically that paired females incorporate much more [3H]-thymidine than females from single-sex infections11. The most densely labelled nuclei were Stage 1 vitellocytes. If mature egglaying females were separated from their paired males, these females had a continual decrease in uptake of [3H]-thymidine compared with females maintained with male partners. This uptake increased again when isolated females were re-mated with males. The results show that the male worm has an effect on DNA synthesis in the vitellocytes of the female and, in addition, that a mitogenic signalling pathway is switched on in the vitellocytes when the female receives the developmental stimulus from the male. http://parasites.trends.com
The intimate and permanent association between male and female worms necessary for reproduction to occur is known to be independent of sperm transfer14. In an attempt to localize the stimulatory factor, male worms were cut into segments and incubated with virgin females that paired in vitro with the male segments15. Vitelline gland development occurred only in that small segment of the female that was in direct physical contact with the male segment. Furthermore, only the portions in contact with cut male segments showed significantly higher uptake of [3H]-thymidine (indicating increased DNA synthesis and cell division) than the non-contacted portions. This shows that each part of the male body along the anterior–posterior axis can stimulate the female and that the female developmental response is not propagated longitudinally. If Schistosoma mansoni females are paired with male partners of a different species, several such heterospecific pairings result in females that, although fertile, produce reduced numbers of eggs16. In addition, they remain smaller than females from homospecific pairs (Fig. 1c,d). Thus, the male stimulus acts to a certain extent in a species-specific manner. A purely tactile stimulus seems to be inconsistent with these observations because it would not be expected to be species specific. It is more likely that the stimulation involves a chemical transfer. Acetone or ether extracts from males could initiate the development of vitellocytes in vitro in females from single-sex infections. It has thus been suggested that lipids might be involved in the transfer of the male signal17. Male extracts appear to enhance the transcription of the major eggshell-coding gene p14 in virgin females18. The expression of this gene under normal conditions occurs in females only when they are paired12. Cholesterol has been shown to be transferred between male and female adult schistosomes19. From these observations, it has been speculated that steroid hormones might be important
Review
Fig. 2. Hypothetical scheme of the differentiation processes in the vitellarium that are induced by pairing with the male. The vitellaria of virgin females contain low numbers of vitellocytes, and these are all undifferentiated Stage 1 cells9. These cells are the target of the male stimulation. From the morphological data, at least two signalling pathways are probably involved: one that induces S phases and a multiplication in number of Stage-1 vitellocytes; and a second that initiates unequal mitotic cell divisions, resulting in daughter cells that differentiate into Stage 2, Stage 3 and finally Stage 4 cells.
TRENDS in Parasitology Vol.17 No.5 May 2001
Male signal
Stage 1
Equal cell division
Mitogenic genes expressed
Stage 1
Unequal cell division
Stage 1
Stage 2 Stage 1
Genes for differentiation expressed Terminal differentiation Stage 3
Stage 4 TRENDS in Parasitology
in male–female interactions20. Recently, a steroidhormone receptor has been identified that binds to promoter elements of the p14 gene21. Previous reports have described the occurrence of ecdysones in adult schistosomes22. However, the peaks of synthetic activity of ecdysone in schistosome development militate against its having role in male–female interaction: the highest concentrations of free ecdysteroid were found in lung-stage worms, not in adults22. Ecdysteroids were originally characterized as the moulting hormones of insects and, following recent taxonomy, are characteristic for a division of protostomes called the Ecdysozoa, which includes nematodes and arthropods. Only these groups exhibit ecdysone-regulated moulting, whereas the metabolic function of ecdysteroid in the other large protostome clade, the Lophotrochozoa (which includes the platyhelminths), is still not clear23. This would call into question a regulatory role for ecdysone in schistosome development; as a consequence, there should be no nuclear ecdysone receptor regulating the effects of ecdysteroids in schistosomes, because this would be considered to be a characteristic marker of the Ecdysozoa24. It might be important for an understanding of the nature of the male stimulus that schistosome females from single-sex infections occasionally develop some differentiated vitelline cells and even some malformed eggs with shells, despite the complete absence of males25. This, however, requires very long incubation times of unisexual females in the final host, and there is considerable variation between populations recovered from different mouse strains. http://parasites.trends.com
229
Nevertheless, these observations demonstrate a limited autonomous ability of vitellocytes to differentiate, although the development of a normal number of vitellocytes requires the permanent stimulus given by the male. There is still another observation with potential significance for understanding the nature of developmental induction stimulated by the male. Homosexual pairings of mature males can induce vitellocyte development up to Stage 4 in virgin males when the virgin male is located in the gynecophoric channel of the adult male partner for long enough26. The vitellocytes appear at the same position as in the female. If one considers this unusual abnormality in a phylogenetic context, it is tempting to interpret this as an evolutionary atavism. Schistosomes evolved from monoecious ancestors and, in most monoecious Digenea, the male reproductive system matures before the female system (protandry), which helps to avoid self-fertilization. In such proterandrous organisms, maturation of the female reproductive system is developmentally repressed as long as the male system is functionally active. Later in development, a de-repression mechanism specifically stimulates the differentiation of female reproductive cells. It is possible that, in recent evolutionary time, the male schistosome as a separate sex has taken over the role of de-repressing developmental events that, in the hermaphroditic ancestors, occurred in late development. The nature of male stimulation in schistosomes differs distinctly from the well-known mechanism of male-controlled egg maturation in Drosophila females27. In this species, a sex peptide is synthesized by the male and transferred to the female during copulation. If sexually mature females do not copulate with males, the production of new eggs is prevented by controlled resorption (apoptosis) of oocytes. The sex peptide in Drosophila controls vitellogenic oocyte maturation at a final stage of oogenesis during the passage of oocytes from Stage 9 to Stage 10, shortly before oviposition. By contrast, in schistosomes, the male signal molecule controls earlier events in vitellocyte differentiation. Signal transduction pathways in the female
How can a chemical signal from the male induce cellular processes in the vitelline gland inside the female body? As a first step, the signal has to cross the surface of the female. Schistosomes are not surrounded by a cuticula but are instead covered by a tegument, which is a cytologically unusual structure. The tegument is a syncytium bounded externally by two lipid bilayer membranes28. Our knowledge of the biochemistry of the schistosome surface is still in its infancy. Rapid internalization of fluorescent lipids and their analogues throughout the membrane has been seen (particularly of the lipid ceramide, which mediates cell signalling in various eukaryotes), suggesting that there is a transport mechanism
230
Acknowledgements I thank Christoph Grevelding and Khalid Zemzoumi (both at the University of Düsseldorf, Germany) for valuable ideas and suggestions. Dedicated to Paul Basch in appreciation of his essential contribution to understanding male–female interactions in schistosomes.
Review
TRENDS in Parasitology Vol.17 No.5 May 2001
through the membrane29. This transport has been observed to be restricted to distinct patches at the surface, showing that the surface of schistosomes is not homogeneous but has specialized microdomains that allow non-polar lipids to pass through. Interestingly, other studies30 have shown that the schistosome surface membrane contains caveolaelike structures – flask-like membrane microdomains that maintain a protein concentration different from that in surrounding membrane. In mammals and other eukaryotes, caveolae are suggested to be centres through which signal-transducing events are focused via a linker protein called caveolin31. In schistosomes, caveolae-like domains are mainly found in the tubercles, which are specialized areas on the dorsal surface of the male. Interestingly, an S. mansoni receptor protein serine/threonine kinase of the transforming growth factor receptor family has been colocalized to the tubercles on the dorsal surface of male worms32. It is thus tempting to speculate that, in schistosomes, as in other organisms, caveolae might also be focal points for receptors involved in signal transduction. However, the serine/threonine kinase receptor in schistosomes is detected only in adult males and is probably not directly related to the male–female interaction processes. Transport of a male signal from the tegument to the vitelline gland would be impeded by the fact that schistosomes, like other platyhelminths (being Parenchymia), are completely filled with parenchyma, lacking any kind of body cavity or circulatory fluid suited for molecular transport. The question thus arises of whether the signal is transported through intercellular spaces or whether it moves across membranes. In Drosophila, the Gurken protein, which is a growth factor that acts as a ligand to a receptor tyrosine kinase, is released from the oocyte and internalized by the follicle cells during
References 1 Popiel, I. and Basch, P.F. (1984) Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments. J. Exp. Zool. 232, 141–150 2 Tchuem Tchuenté, L.A. et al. (1996) Mating behaviour in schistosomes: are paired worms always faithful? Parasitol. Today 12, 231–236 3 Ribeiro-Paes, J.T. and Rodrigues, V. (1997) Sex determination and female reproductive development in the genus Schistosoma: a review. Rev. Inst. Med. Trop. São Paulo 39, 337–344 4 Leutert, R. (1974) Sex differentiation and gametogenesis in Bonellia viridis Rolando. J. Embryol. Exp. Morphol. 32, 169–193 5 Basch, P.F. (1990) Single sex schistosomes and chemical messengers. Reply. Parasitol. Today 6, 298 6 Erasmus, D.A. (1973) A comparative study of the reproductive system of mature, immature and ‘unisexual’ female Schistosoma mansoni. Parasitology 67, 165–183 7 Schüßler, P. et al. (1995) An isoform of ferritin as a component of protein yolk platelets in Schistosoma mansoni. Mol. Reprod. Dev. 41, 325–330 http://parasites.trends.com
signalling33. This is, however, only a bilateral cell–cell interaction of adjacent cells and not a signal transport mechanism that connects different organs with each other. It is noteworthy, however, that most schistosome tissues (and not just the tegument) are syncytial in nature. Syncytial organization might be essential for the movement of signal as well as nutritive molecules34 in organisms that depend on diffusion as a mechanism for substrate transport. Conclusions and future directions
Owing to their importance in cancer research, there are many drugs available that more-or-less specifically inhibit signal-transduction molecules. This opens a way to relate cellular target processes in schistosome reproductive development, such as vitellocyte multiplication or differentiation, with some signalling molecules that might play a role in regulating these processes. cDNA libraries can be screened for the mRNA sequences of these signalling molecules and the clones obtained used for RNA interference to achieve a more specific inhibition than is possible with chemical blockers. When the expression of transgenes in schistosomes (which at present has been applied with some preliminary success35) becomes a reality, very specific investigations of gene function will be possible. As soon as signalling molecules that govern schistosome reproductive development are identified, their partners in the signal transduction cascade should be identified using the yeast two-hybrid system. The identification and inhibition of male-induced signalling molecules in the reproductive organs of female schistosomes might open alternative ways to control schistosomiasis, because this would inhibit egg laying and therefore stop pathogenesis in infected humans. Prevention of egg production would also interrupt the propagation of this parasite.
8 Köster, B. et al. (1988) Identification and localization of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni. Mol. Biochem. Parasitol. 31, 183–198 9 Erasmus, D.A. (1975) Schistosoma mansoni: development of the vitelline cell, its role in drug sequestration, and changes induced by astiban. Exp. Parasitol. 38, 240–256 10 Popiel, I. et al. (1984) The morphology and reproductive status of female Schistosoma mansoni following separation from male worms. Int. J. Parasitol. 14, 183–190 11 Den Hollander, J.E. and Erasmus, D.A. (1984) Schistosoma mansoni: DNA synthesis in males and females from mixed and single-sex infections. Parasitology 88, 463–476 12 Grevelding, C.G. et al. (1997) Female-specific gene expression in Schistosoma mansoni is regulated by pairing. Parasitology 115, 635–640 13 Kunz, W. et al. (1995) Schistosoma mansoni: control of female fertility by the male. Mem. Inst. Oswaldo Cruz 90, 185–189 14 Basch, P.F. and Basch, N. (1984) Intergeneric reproductive stimulation and parthenogenesis in Schistosoma mansoni. Parasitology 89, 369–376 15 Basch, P.F. (1988) Schistosoma mansoni: nucleic
16 17
18
19
20
21
acid synthesis in immature females from singlesex infections, paired in vitro with intact males and male segments. Comp. Biochem. Physiol. B 90, 389–392 Jourdane, J. et al. (1995) Parthenogenesis in Schistosomatidae. Parasitol. Today 11, 427–430 Shaw, J.R. et al. (1977) Schistosoma mansoni: in vitro stimulation of vitelline cell development by extracts of male worms. Exp. Parasitol. 42, 14–20 Rumjanek, F.D. et al. (1987) Genomic regulation in immature females of Schistosoma mansoni. Mem. Inst. Oswaldo Cruz 82 (Suppl. 4), 209–212 Haseeb, M.A. et al. (1985) The uptake, localization and transfer of [4-14C]cholesterol in Schistosoma mansoni males and females maintained in vitro. Comp. Biochem. Physiol. A 82, 421–423 Silveira, A.M.S. et al. (1986) Transfer of [14C] cholesterol and its metabolites between adult male and female worms of Schistosoma mansoni. Comp. Biochem. Physiol. B 85, 851–857 Freebern, W.J. et al. (1999) Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression. J. Biol. Chem. 274, 4577–4585
Review
22 Nirde, P. et al. (1983) Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite Schistosoma mansoni. FEBS Lett. 151, 223–227 23 Knoll, A.H. and Carroll, S.B. (1999) Early animal evolution: emerging views from comparative biology and geology. Science 284, 2129–2137 24 de Mendonca, R.L. et al. (2000) Hormones and nuclear receptors in schistosome development. Parasitol. Today 16, 233–240 25 Shaw, J.R. and Erasmus, D.A. (1981) Schistosoma mansoni: an examination of the reproductive status of females from single sex infections. Parasitology 82, 121–124 26 Armstrong, J.C. (1965) Mating behavior and development of schistosomes in the mouse. J. Parasitol. 51, 605–616 27 Soller, M. et al. (1999) Control of oocyte maturation in sexually mature Drosophila
TRENDS in Parasitology Vol.17 No.5 May 2001
females. Dev. Biol. 208, 337–351 28 Hockley, D.J. and McLaren, D.J. (1973) Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. Int. J. Parasitol. 3, 13–25 29 Redman, C.A. and Kusel, J.R. (1996) Distribution and biophysical properties of fluorescent lipids on the surface of adult Schistosoma mansoni. Parasitology 113, 137–143 30 Racoosin, E.L. et al. (1999) Caveolae-like structures in the surface membrane of Schistosoma mansoni. Mol. Biochem. Parasitol. 104, 285–297 31 Parton, R.G. (1996) Caveolae and caveolins. Curr. Opin. Cell Biol. 8, 542–548 32 Davies, S.J. et al. (1998) A divergent member of the transforming growth factor β receptor family
231
33
34
35
36
from Schistosoma mansoni is expressed on the parasite surface membrane. J. Biol. Chem. 273, 11234–11240 Peri, F. et al. (1999) Local Gurken signaling and dynamic MAPK activation during Drosophila oogenesis. Mech. Dev. 81, 75–88 Skelly, P.J. et al. (1998) Glucose transport and metabolism in mammalian-stage schistosomes. Parasitol. Today 14, 402–406 Davis, R.E. et al. (1999) Transient expression of DNA and RNA in parasitic helminths by using particle bombardment. Proc. Natl. Acad. Sci. U. S. A. 96, 8687–8692 Vogel, H. (1941) Ueber den Einfluss des Geschlechtspartners auf Wachstum und Entwicklung bei Bilharzia mansoni und bei Kreuzpaarungen zwischen verschiedenen Bilharzia-Arten. Zentralbl. Bakteriol. (Abt. I) 148, 78–96
Mass spectrometric strategies: providing structural clues for helminth glycoproteins Stuart M. Haslam, Howard R. Morris and Anne Dell Here we review current knowledge of helminth glycans and introduce parasitologists to the power of the mass spectrometric techniques that have been largely responsible for defining their carbohydrate moieties. A brief overview of glycosylation in other eukaryotes is presented, with a focus on mammalian glycosylation, to facilitate understanding of how parasite structures might be recognized as ‘self’ or ‘foreign’ by the immune system of the host.
Stuart M. Haslam Howard R. Morris Anne Dell* Dept of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK SW7 2AY. *e-mail: [email protected]
Glycoproteins are abundant constituents of the outer surfaces and the excreted–secreted antigens of helminth parasites. Their structurally complex carbohydrate chains are prime candidates for host–parasite recognition events at all stages of parasitism. Indeed, glycoconjugates are increasingly being implicated in immune responses to parasite infections. In terms of their glycobiology, probably the most studied group of parasites is the schistosomes. For example, glycans carrying the Lewisx epitope have been shown to contribute to the known T helper type 1 (Th1) cell downregulation associated with schistosomiasis in both mice1 and humans2, in the induction of host cytotoxic antibodies that lead to the lysis of host cells3–5 and to the induction of hepatic granuloma6,7. However, little is known about the molecular basis of carbohydrate-mediated host–parasite interactions. This is largely because the structures of relatively few helminth carbohydrates have been rigorously defined and even fewer are
available in quantities that permit immunological functional investigations. High-sensitivity mass spectrometry (MS) provides a means of directly addressing the first of these issues and, indirectly, the second; by amassing an increasing number of structurally defined parasite glycans we can compare structural relationships to host molecules and start to predict their potential functionality. N- and O-glycosylation
There are two main types of protein glycosylation: N-glycosylation, in which the glycan is attached to an Asn residue present in the tripeptide consensus sequon Asn-X-Ser/Thr (where X can be any amino acid except Pro), and O-glycosylation, in which the glycan is attached to a Ser or Thr residue (reviewed in Refs 8,9). Glycoproteins can contain just N- or O-glycans, or a combination of both. N-glycans are synthesized from a common precursor (Fig. 1), which is processed by stepwise trimming and stepwise addition of new sugar residues. Trimming by α-glucosidases and α-mannosidases without any subsequent glycosyl addition to the periphery results in glycans having the composition Man5-9GlcNAc2. Glycans of composition Man5-9GlcNAc2 are designated ‘high mannose’ or ‘oligomannose’. In invertebrates, it is not uncommon for additional α-mannose residues to be removed, giving glycans
http://parasites.trends.com 1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0169-4758(00)01860-3