A CTIN IN SPERMATIDS AND SPERMATOZOA OF TELADORSAGIA CIRCUMCINCTA A N D TRICHOSTRONGYLUS COLUBRIFORMIS
(NEMATODA, TRICHOSTRONGYLIDA)
MANSIR A.*, NOURY-SRAÏRI N.*, **, C A B A R E T J.***, K E R B O E U F D.***, ESCALIER D.****, D U R E T T E - D E S S E T M.-C.* & J U S T I N E J.-L.*
S u m m a r y :
Trichostrongyle nematodes provide valuable models for the study of nematode sperm, because their male germ cells are large and elongate, allowing easy identification of cell organelles. A previous study led to the unexpected result that actin co-localizes with MSP (Major Sperm Protein) in spermatids and spermatozoa of Heligmosomoides polygyrus. Actin expression in male germ cells of Teladorsagia circumcincta and Trichostrongylus colubriformis was studied using a monoclonal anti-actin antibody. Actin was demonstrated in the « fibrous bodies » in spermatids and in an anterior cap in spermatozoa. The actin labelling pattern in the two species studied was similar to that found in H. polygyrus, suggesting that this distribution of actin might be general for nematode male germ cells.
KEY WORDS
:
spermatozoon, actin, Nematoda, Teladorsagia circumcincta, Trichostrongylus colubriformis, Heligmosomoides polygyrusRésumé : L'ACTINE DANS LES SPERMATIDES ET LES SPERMATOZOÏDES DE TELADORSAGlA CIRCUMCINCTA ET T R I C H O S T R O N G Y L U S COLUBRIFORMIS (NEMATODA, TRICHOSTRONGYLIDA)
Les nématodes Trichostrongylida sont des modèles intéressants pour l'étude des spermatozoïdes de nématodes, parce que leurs cellules germinales mâles sont allongées et de grande taille, ce qui permet une identification facile des organites. Une étude antérieure a montré de manière inattendue que l'actine a la même localisation que la MSP (Protéine Spermatique Majeure) dans les spermatides et les spermatozoïdes de Heligmosomoides polygyrus.
L'expression de l'actine a été étudiée dans les cellules germinales mâles de Teladorsagia circumcincta et Trichostrongylus
colubriformis en utilisant un anticorps monoclonal anti-actine. La présence d'actine a été démontrée dans les « corps fibreux » des spermatides et dons le capuchon antérieur des spermatozoïdes. Le marquage anti-actine dans les deux espèces étudiées était similaire à celui décrit chez H. polygyrus, ce qui suggère que cette distribution de l'actine pourrait être générale dans les cellules germinales mâles de nématodes.
MOTS CLÉS : spermatozoïde, actine, Nematoda, Teladorsagia circumcincta, Trichostrongylus colubriformis, Heligmosomoides polygyrus
INTRODUCTION
S
p e r m a t o z o a o f n e m a t o d e s are devoid o f a fla- gellum and s h o w a m o e b o i d m o v e m e n t s (refe- r e n c e s in Mansir & Justine, 1 9 9 5 ) . T h e protein associated with this m o v e m e n t is not actin but the n e m a t o d e sperm specific major-sperm-protein or MSP (Theriot, 1 9 9 6 ) . In Caenorhabditis elegans (Nelson, Roberts & Ward, 1 9 8 2 ) and Ascaris (Nelson & Ward,* Laboratoire de Biologie parasitaire-Protistologie-Helminthologie, ERS 156 CNRS, Muséum national d'histoire naturelle, 61, rue Buffon, F 75231 Paris Cedex 05, France.
** Present address : Département halieutique. Institut Agronomique et Vétérinaire Hassan II. HIDAOA, BP 6202, Rabat, Morocco.
*** Station de Pathologie aviaire et Parasitologie, INRA, F 37380 Nou- zilly, France.
**** Laboratoire de Biologie de la Reproduction et du Développe- ment, CHU Bicêtre, F 94275 Le Kremlin-Bicêtre, France.
Correspondence : Jean-Lou Justine.
Tel. : + 33 1 40 79 35 03 - Fax : + 33 1 40 79 34 99 E-mail:justine@mnhn.fr.
Parasite, 1997, 4, 373-376
1 9 8 1 ) , the amount o f actin is very low in spermatozoa.
A recent study (Mansir & Justine, 1 9 9 6 ) has s h o w n that Heligmosomoides polygyrus, a trichostrongyle n e m a - tode, has large spermatids and spermatozoa in w h i c h cell organelles can b e easily identified. In this species, i m m u n o c y t o c h e m i s t r y observations w e r e performed with anti-actin and anti-MSP antibodies, previously s h o w n by immunoblotting to b e strictly specific for their respective targets. A clear, but u n e x p e c t e d , result o f immunocytochemistry was that actin co-localizes with MSP in spermatids and spermatozoa. Moreover, the s a m e localization o f actin in spermatids was also detected b y fluorescent phalloidin, which is specific to F-actin, and the actin-associated protein tropomyosin was detected in spermatozoa in the s a m e region as actin. Comparative studies have therefore b e e n under- taken in two other s p e c i e s o f trichostrongyle nema- todes (Trichostrongylus colubriformis and Teladorsagia circumcincta) with similar sperm morphology. In this paper, w e confirm the results o n actin localisation pre- viously obtained in the male germ cells o f Heligmo- somoides polygyrus by similar results obtained in these t w o s p e c i e s .
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Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1997044373
MANSIR A. ETAL.
MATERIAL AND METHODS
S
p e c i m e n s o f Trichostrongylus colubriformis and Teiadorsagia circumcincta w e r e r e s p e c t i v e l y o b t a i n e d from experimentally infested rabbits and s h e e p . G e r m cells w e r e o b t a i n e d b y dissecting e a c h male in a drop o f P B S ( p h o s p h a t e buffer saline, Sigma) o n a pit slide. T h e genital system was gently pressed to release germ cells, w h i c h adhered to the slide without coating. T h e pits w e r e dried for o n e hour.Previous e x p e r i m e n t s with Heligmosomoides polygyrus have s h o w n that the air-drying m e t h o d o f fixation c a u s e d artefacts at the level o f the nucleus (Mansir &
Justine, 1996), but that determination o f the localiza- tion o f p r o t e i n b y i m m u n o c y t o c h e m i s t r y w a s not affected. Cells w e r e p r o c e s s e d at r o o m temperature, fixed in a c e t o n e for 10 min, and rinsed in PBS ( 3 X 5 m i n ) . Non-specific antigens w e r e b l o c k e d with 2 % B o v i n e Serum Albumin ( S i g m a ) in PBS ( B S A - P B S ) for 45-90 min. A monoclonal anti-actin antibody developed against chicken gizzard actin (Amersham N 3 5 0 ) diluted at 1/100 or 1 / 4 0 0 in BSA-PBS or PBS was applied for 4 0 min. After washing ( P B S 3 x 5 min) the goat anti- m o u s e FITC-conjugated antibody (Nordic, 1/40 in P B S ) w a s applied for 4 0 min and then w a s h e d ( P B S 3 x 5 m i n ) . Counterstaining was performed with Evans B l u e ( 2 °/oo in P B S , 10 m i n ) . After a final wash ( P B S 3 x 5 min), the preparation w a s m o u n t e d in Citifluor (Citifluor Ltd, London, U K ) . Controls w e r e d o n e b y omitting the first antibody; they were negative and thus are not further illustrated or m e n t i o n e d . Observations w e r e made with an O l y m p u s BH-2 epifluorescence microscope. Scanning electron microscope observations o f sperm w e r e performed with a routine m e t h o d o f glutaraldehyde fixation followed b y dehydration in ethanol (Mansir & Justine, 1996).
s h o w e d an intense labelling o f the cytoplasm, c o n s i s - ting o f numerous large dots. Late spermatids w e r e less strongly labelled (Fig. 2 D ) . G e r m cells o f Tr. colubri- formis also s h o w e d different labelling patterns during
s p e r m i o g e n e s i s (Figs. 2 E , F ) . A dot-like pattern o f actin distribution was found in the cytoplasm (Fig. 2 E ) , but spermatozoa s h o w e d an intense labelling o f the cap opposite the nucleus and a w e a k e r labelling o f the cytoplasm (Fig. 2 E ) . In this s p e c i e s , it was possible to label the spermatozoa p a c k e d in the genital tract, in regions w e r e the sheath o f the tract w a s disrupted (Fig. 2 F ) .
DISCUSSION
I
n spermatids o f H. polygyrus, a previous study (Mansir & Justine, 1996) has demonstrated, by the u s e o f electron m i c r o s c o p y and i m m u n o c y t o c h e - mistry, that the actin-containing structures in sperma- tids are the fibrous b o d i e s . Fibrous b o d i e s o f n e m a - tode spermatids are a storage organelle for MSP, from w h i c h MSP is later released in s p e r m a t o z o a to m a k e the cytoskeleton o f the p s e u d o p o d s (Mansir & Justine, 1996; Noury-Sraïri, Gourbault & Justine, 1 9 9 3 ; Ward &Klass, 1 9 8 2 ) . In spermatozoa o f H. polygyrus, the actin labelling, and also the MSP labelling, is restricted to an anterior « c a p », w h i c h is d e v o i d o f organelles (Mansir & Justine, 1996). T h e present study s h o w s that a similar pattern is o b s e r v e d in Tr. colubriformis and
Te. circumcincta.
T h e p r e s e n c e o f actin in n e m a t o d e spermatozoa is o f functional interest. Studies o n С elegans and Ascaris have previously led to the c o n c l u s i o n that the a m o e boid movement o f sperm cells is mediated by MSP (Ita
liano, Roberts, Stewart & Fontana, 1 9 9 6 ; Theriot, 1 9 9 6 ) o w i n g to the q u a s i - a b s e n c e o f actin in mature sper
m a t o z o a (Nelson et al, 1 9 8 2 ; Nelson & Ward, 1 9 8 1 ) . H o w e v e r , actin has b e e n demonstrated in spermatids in several s p e c i e s o f n e m a t o d e s (Mansir & Justine, 1996; Nelson et al, 1 9 8 2 ; Noury-Sraïri étal, 1993). T h e present study demonstrates actin in spermatids o f two further species, thus indicating that previous results (Mansir & J u s t i n e , 1996) o n H. polygyrus m a y b e general for n e m a t o d e sperm cells.
Trichostrongyles are useful m o d e l s for the study o f the cytoskeleton during n e m a t o d e spermiogenesis b e c a u s e they have large s p e r m a t o z o a with antero-posterior polarisation, which allow identification o f organelles better than in C. elegans (Noury-Sraïri et al, 1 9 9 3 ) . H o w e v e r , modification o f spermatozoal m o r p h o l o g y o c c u r s in the female genital tract o f trichostrongyle n e m a t o d e s (Wright & S o m m e r v i l l e , 1 9 8 4 ) and the e x p e r i m e n t s p r e s e n t e d h e r e should b e e x t e n d e d to fully differentiated male germ cells. T h e pattern o f actin
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OBSERVATIONS
T
h e m o r p h o l o g y o f trichostrongyle germ cells is characteristic (Fig. 1). T h e y are e l o n g a t e cells ( 1 5 - 2 0 pm in length, 3-5 pm in width). T h e anterior extremity, or cap, is slightly wider than the rest o f the cell and has a s m o o t h surface. T h e rest o f the cell s h o w s an irregular surface with folds and pits. T h e pyriform nucleus, about 3 pm in length and 1.5 p m in width in its anterior wide pan, is located at the p o s - terior extremity o f the cell. Anti-actin immunolabelling o f Te. circumcincta m a l e g e r m cells (Figs. 2 A - D ) s h o w e d a differential actin distribution d e p e n d i n g u p o n the differentiation stage. Round cells ( s p e r m a t o - cytes) attached to a rachis s h o w e d a general cyto- plasmic labelling (Figs. 2A, B ) . Spermatids, either atta- c h e d to a central rachis (Fig. 2 C ) or free (Fig. 2 D )ACTIN IN NEMATODE MALE GERM CELLS
Fig. 1. — Scanning electron microscopy of a trichostrongyle spermatozoon, Heligmosomoidespolygyrus. C, anterior cap; N, posterior nucleus.
Scale bar 1 pm.
Fig. 2. — Actin labelling of germinal cells in Teladorsagia circumcincta (A-D) and Trichostrongylus colubriformis (E, F). A, B, round cells (probably spermatocytes) attached to rachis (r). C, early spermatids attached to rachis (r). The rachis (r) is counterstained by Evans blue, but this labelling does not correspond to the presence of actin in the rachis. Spermatids are labelled by the anti-actin antibody as dots, corresponding to the fibrous bodies. D, late spermatids and spermatozoa. Early spermatids (es) show a strong homogeneous labelling, late spermatids (Is) are slightly labelled, n, location of nucleus (not labelled). E, late spermatids heavily labelled at the level of the anterior cap (c) opposite the nucleus (n). F, spermatozoa packed in the genital tract, at a level with its sheath disrupted. Only the caps of spermatozoa are labelled. Scale bars 10 pm, for B-F bar in B.
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labelling in the three s p e c i e s studied is similar, thus indicating c o m m o n characteristics o f the cytoskeleton, but s h o w s s o m e species-specific differences, possibly o f t a x o n o m i e interest.
