EXPERIMENTAL

PARASITOLOGY

Schistosoma

42,

14-20

mansoni: Development

(1977)

In Vitro Stimulation by Extracts of Male

J.R. SHAW, I. MARSHALL,~AND Department

of Zoology,

(Accepted

University

for

College, Wales, U.K.

publication

P.O.

D.A. Box

31 August

of Vitelline Worms

Cell

ERASMUS 78, Car&f,

CFZ IXL,

1976)

SAW, J. R., MARSHALL, I., AND ERASMUS, D. A. 1977. Schistosoma munsoni: In vitro stimulation of vitelline cell development by extracts of male worms. Experimental Parasitology 42, 14-20. The iniluence of extracts of male worms on the development of the reproductive system of female Schistosoma mansoni has been investigated under in vitro conditions. Acetone and ether extracts of males initiated the further development of vitelline cells and also produced an increase in the length of females from single sex infections. Extracts of female worms only, “fresh worm” extracts (both male and female worms), “nutrient rich” chemically defined media, and a medium which had supported male worms in vitro prior to the culture of females, all failed to stimulate this development. The data strongly suggest that development of the female reproductive system is dependent to some extent on chemical factors present in the male. INDEX DESCRIPTORS: Schistosoma mansoni: Parasitic helminth; Trematode; Blood fluke; In vitro culture; Male; Somatic growth; Chemical stimulation; Extracts; Development; Vitelline cell; Ultrastructure; Female; Single sex infection; Mouse.

Studies in this laboratory have shown that the reproductive system of females from single sex infections is only partially developed (Erasmus 1973). Furthermore, development of the reproductive system in vitro was dependent on residence by the female in the gynecophoral canal of the male. The presence of sperm in the oviduct of the female was not considered to be the prime factor which initiated maturation (Erasmus 1973; Shaw 1977). This dependence of development upon such a close physical relationship between one male and female schistosomes suggests (a) a physical stimulus, or (b) chemical stimulation involving the translocation of substances between the sexes. Initial, but unsuccessful, attempts to demonstrate the importance of chemical factors have been made in which saline extracts of male

Complete development of the Schistosoma munsoni female reproductive system is dependent upon the presence of male worms. Although a knowledge of the mechanisms responsible for this development is fundamental to our understanding of reproductive biology in schistosomes, the relative importance of tactile stimuli (Armstrong 1965)) nutritional influences (Senft 1968; Vogel 1941), hormonal influences (Moore, Yolles, and Meleney 1954, Sagawa, Ogi, and Sumikoshi 1928; Severinghaus 1928), and insemination or chemicals passed with the sperm (Moore, Yolles, and Meleney 1954; Vogel 1941) has yet to be elucidated. 1 Present address: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, England.

14 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0014-4894

Schistosoma

mansoni:

v~ELLINE

STIMULATION

worms were injected into rabbits (Sagawa, Ogi, and Sumikoshi 1928) and mice (Moore, Yolles, and Meleney 1954) containing only female Schistosoma japonicum and S, mansoni, respectively. The possible existence of chemical stimulation has been reinvestigated by studying the effects of extracts of adult worms on isolated females derived from single sex infections and maintained in vitro. Because these experiments were carried out in vitro the complexities of absorption and alteration of the extracts by host tissues were avoided and direct interaction between the chemicals and parasites was achieved. The response of one female to the extracts was assessedby studying the ultrastructure and histochemical characteristics of the vitelline gland and also by measuring any change in overall length of the body. MATERIALSAND

METHODS

The methods for maintaining Schistosoma mansoni (Puerto Rican strain) in the laboratory and obtaining single sex and mixed infections have been described previously (Erasmus 1973). The culture medium was comprised of human serum/ Earle’s balanced salts (50/50) containing lactalbumin hydrolysate (6500 mg/liter, Gibco-Biocult ), penicillin ( 200 units/ml), and streptomycin (200 PgmJml) sterilized by Millipore filtration. The worms used in the preparation of acetone or ether extracts were recovered from mice by perfusion, washed briefly in citrate saline, sexed, counted, and stored in the selected solvent (1 ml) at -20 C until a sufficient quantity of material had been accumulated. The number of worms used for each extract varied from 18-289. The tissues were homogenized using a Potter Elvehjam Teflon/glass homogenizer, and centrifuged. The solvent was removed from the supernatant in the dark, under reduced pressure and in a stream of nitrogen, and the dried components were reconstituted in 1 ml of solvent which contained 2.5% propylene glycol. The drying process

BY MALE

WORM EXTRACT

15

was repeated and the residual components, in propylene glycol, were incorporated into 2.5 ml aliquots of sterile culture medium. “Fresh worm” extracts were prepared by dissecting paired worms from the mesenteric vessels of mice previously kilIed by cervical dislocation. These worms were removed in this manner instead of by perfusion so that the contents of the gynecophoral canal would be retained, After a brief wash in sterile culture medium the schistosomes were placed in 2.5-ml aliquots of fresh medium, homogenized, and centrifuged, and the supernatants were stored in sealed tubes at -20 C until required. Samples of the culture medium which had previously supported males (100 worms in 2.5 ml for 24 hr) were also obtained and similarly stored. The females pooled from single sex infections and recovered by perfusion from mice 70-100 days postinfection, were washed in three changes of sterile culture medium. Samples of the medium containing extracts were dispensed into 5 x l-cm glass tubes through Swinnex filters (0.22 pm), and three to six females were placed in each vessel. After sealing, the tubes were incubated at 37 C for 4 days, after which the worms were removed and examined. Each experiment, including the controls listed below, was repeated four times, (a) Females were cultured in 2.5 ml of basic medium, as described. (b) Females were cultured without extracts but in medium containing propylene glycol from which the solvent had been removed. (c) Females were cultured in different chemically defined media, i.e., Medium 199, Minimum Essential Medium (Eagle), and Medium NCTC 135 ( Gibco-Biocult Ltd. ) . (d) Females were not cultured but were examined directly after their removal from mice. The extent of the development of females was assessedat the light microscope level by staining for phenolic substances,

16

SHAW,

MARSHALL

AND

TABLE The

Culture

Percentage

medium

of Female Development Mean number worms/extract (range)

Basic medium + acetone or ether extracts of male worms Basic medium + acetone or ether extracts of female worms Basic medium + ‘[fresh worm” extract Basic medium only Medium 199 NCTC 135 Minimum essential medium (Eagle) Noncultured females (70-100 days postinfection)

ERASMUS

I

Schistosoma mansoni which when Exposed to Extracts of

Total number of culture vessels

Exhibited in Vitro Total number of females exposed

150 (18-289)

30

135

88 (15-274)

15

48

110 (50-170) -

8 30 10 10

30 171 24 26

-

10

-

using the fast red B technique (Bell and Smyth 1958), and by measuring the length of the worms. Ultrastructural examination was carried out using the techniques described by Erasmus (1969). RESULTS

Light Microscopy The percentage of Schistosoma mansoni females which produced fast red B-positive material in the region of the vitelline gland is shown in Table I. When the females from single sex infections were exposed to acetone or ether extracts of males, a high proportion (56% ) produced phenolic substances. There was no significant difference between the response of the females exposed to either acetone or ether extracts. However, there was considerable variation between the results obtained in each of the four repeat experiments, the response ranging from 2540%. The response of the females which had been exposed to organic extract of males was gen-

Vitelline

Cell

Total number of females positive with fast red B

76

8

Percentage positive (range per experiment)

56 (25-80)

17

(O-38)

2 28 4 6

6 16 16 23

(O-30) (O-30) (O-23) (O-36)

26

2

8

(O-16)

390

22

6

(O-28)

erally significantly (0.01) greater than that of the females in the control groups. The optimum number of male worms in each extract which resulted in the maximum number of stimulated females was 150-200. The degree of vitelline cell development, demonstrated by the fast red B technique, was also variable (Figs. l-5). Generally a few scattered clusters of stained cells were observed in the posterior two-thirds of the worms. Formed egg shells were observed in 13 (13%) of the females exposed to extracts of males, but these shells only enclosed a few stained vitelline cells (Fig. 6). Compared to the control groups there was a significant (0.01) increase in the length of the females which had been exposed to acetone or ether extracts of males. However, there was some variation within the control groups. In one experiment the mean worm length of the noncultured females was significantly (0.05) greater than that of the females maintained in the basic medium. Table II.

Schistosoma

mznsoni:

VITELLINE

STIMULATION

BY MALE

WORM

EXTRACT

FIG. 1. Photomicrograph of a female Schistosoma mansoni from a singIe sex infection which was stained with fast red B immediately after removal from the mouse. There is no staining reaction alongside the central cecum ( C). X200. FIGS. 2-5. Photomicrographs of Schistosoma mansoni females from single sex infections which were exposed to extracts of male worms in vitro for 4 days. Note the variation in the fast red B staining reaction (V) in the posterior two-thirds of the worms, which indicates vitelline cell development. Fig. 2, X100; Figs. 3 and 4, X450; Fig. 5, X30. FIG. 6. Photomicrograph of a female Schistosomu munsoni from a single sex infection which was exposed to extracts of male wonns in vitro for 4 days. Note the egg shell (E ) with spine (S) but the lack of contents in the egg shell. x450.

18

SHAW,

TABLE

MARSHALL,

II

AND

ERASMUS DISCUSSION

Overall Length of Female Schistosoma

mansoni

Experimental treatment

Number examined

Mean length in millimeters (SW

Noncultured Basic medium only Basic medium + acetone or ether extracts of male worms Basic medium + acetone or ether extracts of female worms

115 119

3.75 (0.06) 3.58 (0.06)

116

4.10 (0.06)

44

3.85 (0.107)

Ultrastructural Studies Groups of four to six cells containing vitelline droplets were observed in the S. mansoni females which had been exposed to acetone or ether extracts of male worms, and the sequence of cell maturation was similar to that described by Erasmus ( 1975). Vitelline cells were most frequently observed in the vitelline duct. Abundant secretory bodies were present in Mehlis’s gland and the necks of the gland cells opened into the ootype lumen. The ovaries of all the females examined failed to exhibit any further development. The tegument of the worms which had been cultured in the basic medium, chemically defined media, or medium to which had been added propylene glycol, remained ultrastructurally unchanged. The tegument of the females which had been exposed to acetone or ether extracts of males, however, exhibited ultrastructural changes from normal. Vacuolation of the basement membrane, formed by the dilation of the basement membrane invaginations, was frequently observed. Similarly, the tegumental cells were also highly vacuolated. Large membrane whorls were present in the tegumental matrix and in the tegumental cells. Muscle layers, nervous tissue, and the excretory system in such females remained essentially unaltered except for the appearance of dense lysosomallike bodies.

The preliminary data presented in this paper strongly suggest that the females from single sex Schistosom mansoni infections were stimulated by acetone- and ether-soluble substances extracted from males. This stimulation resulted in the production of fast red B-positive material in the region of the vitelline gland and ultrastructural studies have confirmed that this response is correlated with the completion of the development of the vitelline cell. It is interesting to note that a small percentage (6%) of the females from single sex infections, which were stained with fast red B immediately after their removal from the host, also exhibited positively stained cells in the region of the vitelline gland. This proportion was increased ( > 16% ) when such females were cultured in vitro for 4 days and indicates that the reproductive status of females in single sex infections is not as precisely defined as previous uItrastructura1 studies have Suggested (Erasmus 1973). This observation also suggests that the deveIopment of a few vitelline cells containing phenolic substances cannot be attributed entirely to the influence of the male. Although it is possible that the observed development of vitelline cells is a response to nutrients contained in the extract material, it is unlikely that the stimulation can be directly or entirely attributable to this factor. The percentage of the females which exhibited development in vitro when cultured in “nutrient-rich” chemically defined media was similar to that of the females cultured in the basic human serum/ Earle’s salts medium. Furthermore, the proportions of the females which contained fast red B-positive material after being exposed to acetone or ether extracts of females or to “fresh worm” extracts were also comparable to that of the females cultured in the basic medium. The response of the females exposed to acetone or ether extracts of males however, was always sig-

Schistosoma

mansoni:

VITELLINE

STIMULATION

BY MALE

WORM

EX~@T

19

nificantly higher than all other categories effective as the gynecophoral canal would studied. help to maintain high level concentrations. Armstrong (1965) suggested that pheroShaw (1977) has commented on the high mone substances produced by the male indegree of contact that exists between the duced somatic development in the female surface of the female and the surface of the and that sexual maturity resulted from male gynecophoral canal of paired worms. tactile stimulation. Previous experiments in It is possible that the gynecophoral canal vitro by one of the present authors (Shaw forms a microenvironment important to the 1977) have shown that residence in the development of the female and that subgynecophoral canal was essential for the stances may be translocated directly from further development of vitelline cells in one tegument to the other. Further work females from single sex infections. It was is in progress to refine the extraction techalso demonstrated that insemination was niques and to elucidate the nature and not an essential prerequisite for this de- functioning of this stimulus. velopment. The present experiments ACKNOWLEDGMENTS strongly suggest that components of male worms stimulated the development of reWe are indebted to the Medical Research Counproductive tissues and this development cil for financial support. We would also like to thank Dr. B, Bevan, Blood Transfusion Service, was accompanied by an increase in worm South Wales, U.K., for generous supplies of serum, length, i.e., somatic development. Thus and Mr. T. W. Davies, Mrs. C. Winters, and Miss these experiments do not enable us to dis- J. Smith for technical assistance. tinguish between a chemical stimuIus associated with somatic development and that REFERENCES involved in the maturation of the reproducARMSTRONG, J. C. 1965. Mating behaviour and detive system. velopment of schistosomes in the mouse. JourThe chemical nature of the stimulating nal of Parasitology 51, 605-616. compound(s) is unknown except that it is BELL, E. J., AND SMYTH, J. D. 1958. Cytological and histochemical criteria for evaluation of desoluble in acetone and ether and thus may velopment of trematodes and pseudophyllidian be associated with the lipid fraction. The cestodes in vivo and in vitro. Parasitology 48, optimum number of extracted males re131-149. quired to stimulate the maximum number ERASMUS, D. A. 1969. Studies on the host paraof females was 150-200, the compounds besite interface of strigeoid trematodes. IV. The ultrastructure of the lappets of Apatemon graing incorporated into 2.5 ml of culture c&s minor> Yamaguti, 1833. Parasitology 59, medium. Thus, the quantity of material 193-201. contained in each male must be small and/ ERASMUS, D. A. 1973. A comparative study of the or the extraction procedures inefficient. The reproductive system of mature, immature and data obtained from females which had unisexual female Schistosoma mansoni. Parasitology 67,165-183. been cultured in medium containing ERASMUS, D. A. 1975. Schistosoma mansoni: De“fresh worm” extract (i.e., including the velopment of the vitelline cell, its role in drug contents of the gynecophoral canal) or in sequestration, and changes induced by Astiban. medium which had previously supported Experimental Parasitology 38, 240-256. K., AND MELENEY,K. E. males suggests that the substance(s) in- MOORE, D.V., YOLLES,T. 1954. The relationship of male worms to the volved is not being released by unpaired sexual development of female Schistosomu manmales in vitro or that the threshold concensoni. Journal of Parasitology 40, 166-185. Y. 1928. trations necessary for stimulation are not SAGAWA, E., OGI, K., AND SUMIKOSHI, Studies concerning the influence of sex on the being reached under the in vitro conditions growth of animal bodies. First report: Experiof the experiments. In paired Schistosoma ments on Schistosoma mansoni. Transactions of mansoni worms minute quantities might be the Japanese Pathological Society l&494-500.

20

SHAW,

MARSHALL

SEVERINGHAUS, A. E. 1928. Sex studies on Schistosoma japonicum. Quarterly Journal of Microscopical Science 71, 653-702. SENFT, A. W. 1968. Studies in proline metabolism by Schistosoma mansoni. I. Radioautography following in vitro errposure to radio-proline ‘“C. Comparative Biochemistry and Physiology 27,

251-261. SHAW, vitro

R. 1977. Schistosoma mansoni: An in study of the factors stimulating the re-

J.

AND

ERASMUS

productive development of females from single sex infections. Experimental Parasitology, in press. VOGEL, H. 1941. Uber den Einfhrss des Geschlechtspartners auf Wachstum und Entwickhmg bei Bilharzia mansoni und Kreuzpaarungen zwischen verschniedenen Bilharzia-Arten. Zentralblatt Fuer Bakteriologie Parasite&&e Abt. I 148,78-96.

Schistosoma mansoni: in vitro stimulation of vitelline cell development by extracts of male worms.

EXPERIMENTAL PARASITOLOGY Schistosoma 42, 14-20 mansoni: Development (1977) In Vitro Stimulation by Extracts of Male J.R. SHAW, I. MARSHALL,~A...
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