Electron microscope study of the blood—testis barrier in an insect: Locusta migratoria

JOURNAL OF ULTRASTRUCTURE RESEARCH

59, 158-172 (1977)

Electron Microscope Study of the Blood-Testis Barrier in an Insect:

Locusta migratoria ANNETTE SZOLLOSI AND CHRISTIANE MARCAILLOU Laboratoire d'Histophysiologie des Insectes, Universitd Pierre et Marie Curie, 12 rue Cuvier, 75005 Paris, France Received October 14, 1976 The locust testis is composed of follicles in which differentiation of the germ line proceeds linearly along the major axis from the apex to the base. By using electron-opaque tracers such as horseradish peroxidase and lanthanum chloride, we demonstrated that the follicle is constituted of two distinct compartments: the apical and basal compartments. The apical compartment containing the young germ cells is readily penetrated by the tracers while the basal one, containing the differentiating germ cells, is tightly closed. In the apical region the tracers permeate the intercellular spaces of the somatic envelopes, perifollicular cell layer and cyst envelopes, and reach the most internal germ cells. In the basal compartment, a new tissue develops under the perifbllicular layer: the inner parietal cell layer. This tissue is constituted of cells displaying long, overlapping processes forming a complex network of intercellular spaces along which extensive septate junctions are found. The tracers permeate the perifollicular cell layer and enter the inner parietal layer but they are stopped before they can traverse it fully. They do not cross the totality of the septate junctions which appear, therefore, to be the main structures restricting the entry of the tracers. Thus, an effective hemolymph-germ cell barrier is actually formed. This type of "blood-testis barrier" can be compared with that found in the mammalian seminiferous tubule: in both cases, a specific environment is provided in which the germ cells complete their development. Also in both cases, the germ cells entering the tight compartment are spermatocytes in early meiotic prophase.

specialized junctions between Sertoli cells divide the germinal epithelium into a ~basal" compartment which contains the spermatogonia and is penetrated by the tracers and an "adluminar' compartment which the tracers never reach and where the germ cells complete their development. In a preliminary note (16), we reported that a blood-testis barrier exists also in an insect: the locust, Locusta migratoria. By using tracers such as iron saccharate, iron gluconate and horseradish peroxidase and with light microscopy, we demonstrated that in Locusta, a testicular follicle is made of an "apical" compartment which contains the youngest germ cells and is readily penetrated by tracers and of a '~basal" one where the spermatids differentiate and which is tightly closed. In the present paper, the penetration of the tracers is studied at the ultrastruc-

The concept of a blood-testis barrier in mammals arose at the beginning of the century (2, 21) from the observation that dyes injected into the blood stained many tissues but did not enter the seminiferous tubules. These observations remained almost forgotten until the methodic experiments of Kormano (14) who used a variety of injected substances, and until the studies of the physiologists who found that there were striking differences in the composition of blood plasma and testicular lymph when compared with the fluid inside the seminiferous tubules or the rete testis [for a review, see Setchell and Waites, (23)] and (12). The physiological data suggested that the "barrier" was located in the seminiferous tubule itself and this was further demonstrated by examination of testes of animals injected with electron-opaque tracers of various size (4, 5). From these studies, it appeared that 158 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0022-5320

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INSECT BLOOD-TESTIS BARRIER

tural level and morphological structures are described which may account for restricted entry of the markers into the basal compartment. MATERIALS AND METHODS The locusts were m a i n t a i n e d in 60 liter wooden cages h e a t e d a n d lit~ internally for 12 h r each day. This supplied a day t e m p e r a t u r e gradient of 32 to 36°C and a n i g h t t e m p e r a t u r e decreasing to 20°C. U n d e r these conditions, the length of the V t h i n s t a r was 9 days. All experiments were performed on 9-day-old V t h i n s t a r larvae and 2-day-old adults. Groups of about 10 follicles were dissected from the anterior region of the testis of each a n i m a l a n d transferred into i ml of either culture medium or saline (16). The tracers used were horseradish peroxidase (HRP) and l a n t h a n u m chloride. Peroxidase (Sigma, type II) was added to the culture medium (pH 7.1; 350 mOsm) a t the concentration of 7.5 mg/ml. The follicles were left from 10 rain to 6 h r in the medium and t h e n fixed for 40 min at room t e m p e r a t u r e in a mixture of 2% glutaraldehyde and 0.1% paraformaldehyde in 0.2 M cacodylate buffer (pH 7.4). The tracer was revealed by incubating either half-follicles or 75 ~ m thick sections obtained with a tissue sectioner for 45 m i n at 37°C in diaminobenzidine (9). Controls for the reaction procedures were incubated either without H~O2 or DAB and were negative. For assay of endogenous peroxidase activity, the technique of Novikoff et al. (20) was used and showed t h a t endogenous peroxidase could in no place be confused with HRP. The tissues were postosmicated in a 2% solution of osmium tetroxide, dehydrated in ethanol a n d embedded in Araldite-Epon. Thick sections of 2 t~m were observed directly with light microscopy and t h i n sections contrasted by lead citrate for electron microscopy. In experiments with l a n t h a n u m , the follicles were placed in a phosphate-free saline (pH 7.1; 320 mOsm) to which l a n t h a n u m chloride (Merck) was added a t the concentration of 20 mM (5 mg/ml). The incubations were carried out for 30 min, the controls being left in the saline without l a n t h a n u m chloride. The follicles were t h e n fixed in glutaraldehyde 1.5% in Dulbecco's phosphate buffered saline (oxo~d) and postosmicated in the same buffer. They were t h e n washed thoroughly in 30% ethanol before being processed as usual. Thin sections were contrasted by lead citrate. For conventional e x a m i n a t i o n of fine structural features, the follicles were either fixed with the glutaraldehyde-paraformaldehyde mixture as described above, postosmicated and t h e n stained in block with uranyl acetate [after Terzakis (25)] or fixed in glutaraldehyde containing CaC12 (5 mM).

RESULTS

I. General Anatomy of the Testis and of the Follicle The male gonads of L. migratoria appear fused in a single ovoid organ although they consist of two series of about 150-200 follicles which open individually into two ventral parallel spermiducts through ductuli efferentes. The numerous follicles are held together by fat-body and by a network of tracheae which penetrate between the follicles but never enter them. Each follicle has the shape of a club and is limited by a continuous envelope. The germ cells develop inside cysts and therefore at least two envelopes separate them from the surrounding hemolymph. From the rounded apex to the tapering base, the succession of the cysts corresponds to the successive stages of development of the germ cells: differentiation and maturation proceed along the major axis of the follicle. The apical region contains dividing gonia and spermatocyte I in early prophase. At the very distal end, a single, large, centrally located apical cell is surrounded by radially oriented gonia. The base of the follicle is occupied by meiotic cells, spermatocytes II and differentiating spermatids. All these stages of course are not yet formed in the young larvae and the relative size of the ~base" increases with the age of the animals (Fig. 1). In the animals studied (larvae in late Vth instar and young adult), the base may also be defined as the compartment where tracers do not penetrate and the apex as the "open" compartment (16) (Fig. 2).

2. Ultrastructural Study of the Somatic Envelopes of the Gonad a. The perifollicular envelope. This envelope consists of a layer of flat perifollicular cells enclosed between an external and an internal lamella, both composed of carbohydrate-rich amorphous material and containing collagen fibrils (Fig. 3). The perifollicular cells possess a cen-

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gonial mitosis

meiosis

spermiogenesis

FIG. 1. Schematic representation of the locust follicle.

b. The inner parietal cell layer. The thickness of the perifollicular cell layer decreases from the apex to the base where under the internal acellular lamella, another tissue appears, made of cells displaying extremely thin overlapping cytoplasmic processes and thus forming a tortuous system of intercellular spaces (Figs. 7, 8, 9, 10). This "inner parietal cell layer" is already present as a thin layer in the region of the meiotic cells. It becomes conspicuous at the level of the young, round spermatids and, from this region to the ductulus efferens, it is visible even with the light microscope. However, the thickness of this layer is highly variable at a given level, fluctuating between 0.5 and 4 t~m. In the young adult a space separates the inner parietal cell layer from the subjacent cyst envelopes. This space later provides the route for the mature bundles of spermatozoa in their descent towards the spermiducts (Fig. 7). In contrast to the perifollicular cells, the interdigitating cells of the inner tissue are attached to one another by some spot desmosomes and gap junctions but essentially by extensive septate junctions (Figs. 8, 9, 10, 11). Septate junctions are cross sectioned only in transverse sections of the follicle, which seems to indicate that the plane of most of the septae is oriented parallel to the long axis of the follicle.

trally located nucleus of elliptic profile and contain the usual organelles: Golgi, rough ER, lysosomes and mitochondria together with some lipid globules. The cisterns of rough ER are often located very close to the plasma membrane and quite often its membrane facing the plasma membrane is not studded with ribosomes (Figs. 3 and 4). The plasma membrane in m a n y places invaginates into pinocytotic vesicles (Fig. 6). A prominent and distinctive feature of these perifollicular cells is their unusually high content of microtubules circularly oriented in relation to the major axis of the 3. Penetration of the Tracers in the Follicle follicle (Fig. 5). Curiously, this orientation is also parallel to that of the collagen fiAll along the follicle, the intercellular brils found in the two acellular lamellae spaces of the perifollicular layer provide (Fig. 6). Periodicity in these fibrils can be open channels through which the tracers observed only in cross sections of the folli- freely reach the internal lamella (Fig. 12). cle. The perifollicular cells differ in size Peroxidase is strongly retained by the according to the region of the follicle. They amorphous substance constituting the two are thicker at the apex and thinner to- acellular lamellae while lanthanum is not wards the base where they seem to be (Fig. 14); however, the two tracers persomewhat stretched. In contrast to these meate the intercellular spaces of the perivariations in thickness, the spaces be- follicular layer in similar ways and only tween them are of constant width of about slight quantitative differences were ob20 n m (Fig. 4). Gap junctions are ex- served in peroxidase treatment related to tremely rare. the length of incubation.

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/ C

A

Fro. 2. Follicle incubated in horseradish peroxidase. (A) Longitudinal section (x 110). (B) Cross section through the apical compartment and (C) cross section through the basal compartment. Two cysts in cross section C are penetrated by the tracer (*). The two others contain more advanced germ cells and are tight. (× 500). • I n the apical c o m p a r t m e n t , tracers h a v e i m p r e g n a t e d the acellular layer, t h e y infiltrate spaces s e p a r a t i n g adjacent cysts

once the internal into the (Fig. 13)

and p e n e t r a t e into t h e m , even t h o u g h some spot desmosomes are present in the envelopes of the cysts. T h e y finally r e a c h the g e r m cells themselves, inclusive of

FIa. 3. Two adjacent cells of the perifollicular cell layer are seen enclosed between the external (EL) and the internal lamella (IL). The latter lines the cyst envelope (Cy). N, nucleus; M, mitochondria; ER, cisterns of endoplasmic reticulum; L, lipid droplet, x 20 000. FIG. 4. Abutment of two cells of the perifollicular layer. Note t h a t the intercellular space is of an almost uniform width throughout. The cells contain numerous microtubules seen in cross section because the follicle has been longitudinally sectioned. The outermost membrane of the ER cisterns close to the plasma membrane (arrows) are devoid of ribosomes, x 50 000. FIG. 5. This region of a perifollicular cell is particularly rich in microtubules on the left side of the cell. Numerous dots are observed which nature has not yet been determined, x 35 000. 162

INSECT BLOOD-TESTIS BARRIER

those located in the center of the follicle (Figs. 2 and 16). The young germ cells are thus quite rapidly exposed to the tracers which penetrate the narrow intercellular clefts separating them as well as the larger spaces found in the regions of the ~ringchannels" (Fig. 17). In some occasions, peroxidase was observed penetrating into cytoplasm (Fig. 15) but this occurs irregularly and is probably an artefact. Somatic cells constituting the cyst envelope and germ cells show endocytosis and capture some tracer i n small phagocytotic vesicles (Figs. 16 and 17). In the basal compartment, the tracers have free access to the inner parietal layer. They penetrate into this layer for some distance before being stopped (Figs. 18 and 19). In spite of great variations in its thickness, this cell layer contains everywhere an intricate and complex network of intercellular spaces. The tracers are found here and there in these spaces and also in the septate junctions. The penetration of markers is specially visible at the periphery of the layer under the perifollicular envelope (Fig. 19) but is also seen in places in the depth of the tissue. In the septate junctions, several interseptae are filled with the tracers which however appear blocked before they can cross the total length of the junctions. On rare occasions, tracers which have reached the internal side of the parietal layer are seen in contact with the cyst cells. In such cases, they never penetrate the cysts and never reach the germ cells. The border between the open and the tight compartment is located in the region where the germ cells are in meiotic prophase (Fig. 20). DISCUSSION

The incubation method used in these experiments was made necessary because of the extensive uptake by various tissues of the insect of any particles injected in vivo (16). The pericardial cells and the hematopoietic organ are able to sequester

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large amounts of any tracer administered, and this process, not only decreases the access of the tracers to the testes but also interferes with the general metabolism and the health of the animals (11, 16). On the other hand, with light microscopy it was difficult to evaluate the adequacy of the technique of incubations. In our view, the present examinations with electron microscopy fully justifies this method since the fine structure of the organ is well preserved even after long treatment (6 hr); besides, the incubation time can be greatly reduced without leading to qualitative changes in the results. It is evident, however, that the penetration of tracers under these experimental conditions is in no way a direct image of what really happens in vivo; it only indicates that there is apossibility for entry of large molecules into certain parts of the gonad and a restriction of that entry in other parts. The two electron-opaque tracers, although different in nature, are difficult to compare by size; in contrast to peroxidase whose molecular weight (40 000) and diameter (60 /~) are known, the ionic lanthanum is a charged molecule which binds to various substances in the intercellular spaces and whose actual size is therefore impossible to evaluate. It is, however, currently considered as a ~small" tracer. In our study both tracers behave similarly: they move through intercellular spaces and, when they are found in cells, they lie within small phagocytotic vesicles. Peroxidase was observed diffusing into the cytoplasm on some occasions but this might be explained by membrane disruption during the experimental procedure. The resulting morphology was then comparable to that described by Wilson et al. (26) who used Friend's adjuvant together with peroxidase. They suggested that the adjuvant might have altered the membrane permeability. In animals used in the present experiments, there is evidently no restriction in penetration of the tracers at the apex of

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INSECT BLOOD-TESTIS BARRIER the g o n a d a n d the g e r m cells a r e directly exposed to t h e s u r r o u n d i n g milieu. A n exp l a n a t i o n for r e s t r i c t e d e n t r y of t h e t r a c e r s in t h e b a s a l c o m p a r t m e n t is complex. As described above, e v e n in this region the perifollicular cell l a y e r is in no w a y a barr i e r a n d it does not oppose the e n t r y of the m a r k e r s w h i c h p e n e t r a t e into t h e i n n e r p a r i e t a l cell layer. A t t e n t i o n w a s t h e r e f o r e paid to the possible routes of e n t r y this tissue m a y provide to the t r a c e r s a n d to the possible g a t e s of, or b a r r i e r , to this entry. We found in this tissue: 1. A highly complex n e t w o r k of intercell u l a r spaces. 2. S o m e r a r e spot d e s m o s o m e s a n d gap junctions. 3. E x t e n s i v e s e p t a t e junctions. It h a s b e e n considered t h a t a t o r t u o u s p a t t e r n of n a r r o w i n t e r c e l l u l a r spaces m i g h t by itself provide a r e s t r i c t i v e b a r r i e r w h e n the distance the t r a c e r s h a v e to t r a v e l is t r e m e n d o u s l y i n c r e a s e d by cellular folds (15). This does not s e e m to hold t r u e in the locust testis b e c a u s e the t r a c e r s which r e a c h the center of the follicle in the apical c o m p a r t m e n t h a v e also to t r a v e l in v e r y long i n t e r c e l l u l a r c h a n n e l s a n d t h e y n e v e r t h e l e s s r e a c h the c e n t r a l region quite rapidly. T h e spot d e s m o s o m e s , a l t h o u g h r a r e r in insects t h a n in v e r t e b r a t e s , h a v e b e e n r e p o r t e d b y s e v e r a l a u t h o r s (22) a n d in t h e g e n i t a l t r a c t b y one of us (24). In no w a y can t h e y oppose the e n t r y of tracers. Only t r u e t i g h t j u n c t i o n s are able to prev e n t p a s s a g e of t r a c e r s applied f r o m e i t h e r side of the junction, b u t until now t h e y h a v e n e v e r b e e n found in i n v e r t e b r a t e s (22). G a p j u n c t i o n s are found occasionally

165

b u t t h e y c a n n o t restrict p a s s a g e of molecules. T h u s we h a v e to t u r n to the s e p t a t e junctions as p o t e n t i a l b a r r i e r s . S e p t a t e junctions h a v e b e e n claimed to be p e r m e a ble to l a n t h a n u m and r u t h e n i u m red Ireviewed by S a t i r and Gilula (22)] b u t some authors have attributed barrier properties to t h e m (10). H a n d a n d Gobel (10) h a v e noted t h a t w h e n the t r a c e r s are added into the fixative, t h e y m a y bind to or s t a i n specific i n t e r c e l l u l a r s u b s t a n c e s a n d t h e i r presence w i t h i n s e p t a t e j u n c t i o n s in these e x p e r i m e n t a l conditions do not demons t r a t e a free p e n e t r a t i o n t h r o u g h the junction. In our e x p e r i m e n t s t h e t r a c e r s were given before fixation. We a c t u a l l y observed peroxidase as well as l a n t h a n u m inside the lattice-like s t r u c t u r e of the s e p t a t e j u n c t i o n s b u t not all i n t e r s e p t a l spaces were filled w i t h the t r a c e r molecules a n d t h e s e molecules did not cross the entire l e n g t h of the junction. Thus, in the case of the testis, it s e e m s to us t h a t the septate junctions, p r e s e n t only in the b a s a l c o m p a r t m e n t , do a c t u a l l y restrict t h e ent r y of the t r a c e r s into this c o m p a r t m e n t . W h e t h e r this m e c h a n i c a l b a r r i e r alone can explain the t i g h t n e s s of the c o m p a r t m e n t is not known, however, a n d o t h e r physiological mechanisms controlling p e r m e a b i l i t y h a v e to be f u r t h e r investigated. The blood-testis b a r r i e r in insects depends on a c o m p l e t e l y different morphol o g i c a l s t r u c t u r e t h a n t h a t of m a m m a l s . The s e m i n i f e r o u s t u b u l e of m a n y s m a l l l a b o r a t o r y m a m m a l s (rat, mouse, g u i n e a pig) is limited by a single l a y e r of "myoid" cells enclosed b e t w e e n two l a y e r s of pro-

FIG. 6. On this transverse section of the follicle one can observe a parallel orientation of the collagen fibrils in the lamellae and of the microtubules (Mr) in the perifollicular cell. The arrow points to a pinocytotic vesicle. IPL, inner parietal cell layer. × 32 000. Fro. 7. Typical aspect at low magnification of the follicular wall in the basal compartment: the perifellicular cell layer (PF) enclosed between the external and the internal lamellae is thin; it is lined by the inner parietal cell layer (IPL) which is itself separated from the cyst envelope (Cy) by a large space containing a flocculent substance (*). This space provides the route through which the mature sperm will later reach the canaliculus efferens. The cyst observed on this micrograph contains young spermatids (Sd). × 16 000. Fro. 8. The cells in the inner parietal layer are strongly interdigitated and linked by extensive septate juncti~)ns. The space between this layer and the cyst envelope is well developed (*). x 40 000.

Fro. 9. In this region, the space between the i n n e r parietal cell layer and the cyst envelope is quite reduced. Septate junctions in the i n n e r parietal layer (arrow) can be seen close to a cyst containing developing spermatid (Sd). x 32 000. Fio. 10. Septate junction in the inner parietal layer. This is a transverse section of the follicle in which most of the septae are also cross sectioned, x 60 000. Fin. 11. Tangential section of the septate junction in the i n n e r parietal layer, x 80 000. Fro. 12. P e n e t r a t i o n of horseradish peroxidase in the intercellular clefts of the perifollicular cell layer (PF). Note t h a t the tracer strongly impregnates the external and internal lamellae and is found in small pinocytotic vesicles of the perifollicular cells (arrows). x 17 000. FIa., 13. In the apical compartment, peroxidase penetrates between cyst cells (Cy) and is found in direct contact with the germ cells (G). x 20 000. FIG. 14. L a n t h a n u m does not react with the external and i n t e r n a l lamellae (arrows). In the apical compartment it penetrates t h r o u g h the cyst envelope (Cy) and reaches the germ cells (G). x 14 000. 166

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FIG. 15. Peroxidase can occasionally p e n e t r a t e into the cytoplasm. This section shows young gonia containing some tracer. This aspect is not constant. The sex vesicle (X chromosome) (X) is clearly recognizable in the nuclei of t h e gonia, x 7000. FIG. 16. In the apical c o m p a r t m e n t , t h e tracer is found even a r o u n d t h e m o s t i n t e r n a l g e r m cells, It enters also small pinocytotic vesicles, x 11 000. FIa. 17. In t h e s a m e region, peroxidase s u r r o u n d s a n intercellular bridge (IB) between two y o u n g gonia. The arrow points to a pinocytotic vesicle of t h e y o u n g g e r m cell. x 20 000. 168

FIG. 18. Penetration of l a n t h a n u m into the basal compartment. This tracer crosses the perifollicular cell layer (PF) and reaches the intricate network of intercellular spaces in the inner parietal cell layer (IPL). It does not reach the innermost side of this layer and does not enter the sperm channel (*). x 27 000. FIG. 19. Penetration of peroxidase into the basal compartment. In contrast to lanthanum, peroxidase impregnates the external and internal lamellae before it enters into the inner parietal layer. There, it penetrates for some distance between the cells but its progression in the tissues is blocked (arrows). It does not reach the internal side of this layer, x 38 000. 169

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FIG. 20. Region of transition between the apical %pen" compartment (upper left) and the basal tight compartment (lower right). The germ cells in this transitional region are spermatocytes in early prophase. The lower cyst containing the two older cells is in contact with the tracer by its external surface but does not allow entrance of the tracer towards the germ cells (arrows). × 6500. tein-polysaccharide m a t e r i a l corresponding to the PAS positive ~'basement memb r a n e " seen with the light microscope. This l a y e r is v e r y similar to the perifollicular cell l a y e r of the locust testis except for the following aspects: while the myo~'d cells of m a m m a l s are filled with microfilam e n t s (4, 5) and are contractile, the perifollicular cells of the locust are filled with microtubules and contractions h a v e n e v e r been observed. The other difference, imp o r t a n t in r e g a r d to the b a r r i e r problem, is t h a t t i g h t junctions b e t w e e n adjacent myo~d cells exclude e n t r y of the tracers in the major p a r t of the seminiferous tubule while such junctions do not occur in the

locust at least not in the y o u n g adult. In regions w h e r e the tracers can p e n e t r a t e t h r o u g h the myo~d layer of the m a m m a lian tubule (4) (15% of the t u b u l e surface), t h e y m e e t a second and more i m p o r t a n t b a r r i e r to f u r t h e r penetration. This barrier is constituted by a peculiar type of occluding junctions: the Sertoli junctions. Those were first described by B r S k e l m a n , in 1963 (3) and h a v e been widely studied since t h e n (1, 4, 5, 7, 8, 18, 19). In t h i n sections, the Sertoli junction appears as a series of focal t i g h t junctions. N e g a t i v e staining and f r e e z e - f r a c t u r e p r e p a r a t i o n s show t h a t t h e y contain up to 40 ridges or rows of particles, parallel to each other,

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which seal the intercellular spaces efficiently. The septate junctions found in the locust follicle seem to play the same role as these Sertoli junctions in the mammalian tubule and the basal compartment of the locust follicle may be regarded as homologous to the adluminal compartment of the seminiferous tubule (Fig. 21). In both cases, the barrier provides a tight compartment in which some meiotic and the postmeiotic processes take place. In maremals, the way how the germ cells pass fi'om the basal to the adluminal compartment has not been precisely demonstrated and, in the same way, what happens in the locust follicle when a developing cyst moves as a whole from the apical patent compartment to the basal tight one is not fully understood. Septate junctions in the inner parietal layer explain how the entry

of tracers is restricted from the outside but why do not apical tracers leak into the basal compartment from the inside? it is true that some septate junctions develop between adjacent aging cysts but they are rare and they cannot alone explain that fact. Most probably, parallel to the evolution of the germ cells, the somatic cells forming the cyst envelopes also become modified. Development of new junctional specializations would be only one aspect of such modifications which need further investigations. The exact stage at which a germ cell passes from the open to the closed compartment is difficult to define. In the case of the locust follicle, the border between the two compartments seems to be located in the region of spermatocytes in early prophase. This probably means that the

permeable compartment

]----]

tight compartment

basal compartment

1 LOCUSTA FOLLICLE

adluminal ;ompartment

MAMMAL

TUBULE

FIG. 21. Schematic representation of the blood testis barrier in the locust and in a mammal. In both cases, the young germ cells divide in a compartment which is permeable to substances present in the blood but they complete their development in a tight compartment. The arrows indicate the orientation of the germ line differentiation.

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SZOLLOSI AND MARCAILLOU

barrier is established as soon as the first germ cells reach this stage (17). Mammalian spermatocytes which enter the tight compartment are also spermatocytes in leptotene and the barrier is established when the first germ cells enter meiosis (6). It has been widely accepted that an important role of the blood-testis barrier in mammals is to provide a very special microenvironment for the germ cells to develop. Analysis of the tubule fluid has shown that some components are either absent or highly concentrated in it (23). Such physiological studies have not yet been performed in insects and the composition of the fluid in which the sperm differentiate is unknown. It is therefore premature to speculate on the physiological significance of a blood-testis barrier in insects; one can only tentatively assume that this barrier may represent a protective mechanism for spermiogenesis which is a highly sensitive process. On the other hand, the existence of the barrier described in this study does not contradict the hypothesis of Kambysellis and Williams (13) who postulated that some macromolecules from the blood must enter the testis to promote meiosis and further development of the germ cells in diapausing Saturnids. If this turns out to be a general feature in insects, then it may be worth noting that in the locust the pre-meiotic stages are located in the apical compartment which is actually an open compartment. This work has been carried out in the ~Equipe de Recherche Associ~e au CNRS, E.R.A. n°570 ''. The electron microscopical observations have been made in the "Laboratoire de Microscopie Electronique appliqude h la Biologie, CNRS" and in the "Laboratoire de Physiologie du Ddveloppement du Coll6ge de France et de rUniversit6 Pierre et Marie Curie."

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