MICROSCOPY RESEARCH AND TECHNIQUE 20:43-49 (1992)
Effects of Vitamin A Deficiency on the Inter-Sertoli Cell Tight Junctions and on the Germ Cell Population NERMINE ISMAIL AND CARLOS R. MORALES Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2
KEY WORDS
Vitamin A deficiency, Blood-testis barrier, Seminiferous epithelium
ABSTRACT When 20-day-old rats are placed on a vitamin A deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells, a few residual AO,A, spermatogonia, and preleptotene spermatocytes (PL). The type A, spermatogonia and PL spermatocytes are arrested in their G, phase. In VAD rats type A,-A,, intermediate (In) and B spermatogonia and all types of spermatocytes (except PL spermatocytes) and spermatids are eliminated from the seminiferous tubules. Two questions were raised in this investigation: 1)Is there, in VAD rats, any correlation between a breakdown of the blood-testis barrier (e.g., Sertoli cell tight junctions) and germ cell loss? 2) Is the disappearance of most germinal cells due to their degeneration during spermatogenesis or to a maturation depletion process resulting from an arrest of spermatogenesis at the spermatogonial stage? To investigate these questions four groups of male SpragueDawley rats (20-days old) were fed a VAD diet for 7 to 12 weeks. The testes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate containing 2% lanthanum nitrate, an electron opaque tracer used to test the patency of Sertoli cell tight junctions. The lanthanum permeated the intercellular space of the basal compartment but was arrested by normal inter-Sertoli cell tight junctions. The seminiferous epithelium showed numerous degenerating germ cells, some being internalized by Sertoli cells as membrane-bound phagosomes. Thus, these results indicate firstly that inter-Sertoli cell tight junctions remain intact during vitamin A deficiency, and secondly that in a first phase nonviable germinal cells degenerate during spermatogenesis and their residues are actively phagocytosed by Sertoli cells followed by a second phase where the regressed state of the seminiferous epithelium is maintained by a maturation depletion condition resulting from an arrest of spermatogonial proliferation and differentation.
INTRODUCTION
toli cell tight junctions forming the blood-testis barrier. As a consequence, the modified milieu of the adluminal When 20-day-old rats are placed on a vitamin A de- compartment would not permit the normal differentificient diet (VAD) for a period of 12 weeks, the semi- ation of the spermatocytes and spermatids that would niferous tubules are found to contain only Sertoli cells, then degenerate. a few type A spermatogonia, and preleptotene sperAnother question raised by the regression of the sematocytes (Huang and Hembree, 1979; Morales and miniferous tubules in VAD rats was whether the deGriswold, 1987; Ismail et al., 1990). The type A sper- pletion of the germ cells from the epithelium was the matogonia, of the type A, class, are seemingly arrested consequence of the degeneration of the germinal cells in their G2phase of the cell cycle, whereas type A2-A4, that were in the process of differentiating into spermaintermediate, and type B spermatogonia and all types tozoa or of a maturation depletion process resulting of spermatocytes except a few preleptotene spermato- from the arrest of the proliferation of spermatogonia cytes are eliminated from the seminiferous epithelium with the consequence of a progressive disappearance (Ismail et al., 1990). Following this regression, sper- with time of the more advanced germinal cells from the matogenesis can be reinitiated by the administration of seminiferous epithelium or even of a combination of retinol but not by its metabolic derivative, retinoic acid these two processess. (Howell et al., 1963; Ahluwalia and Bieri, 1971; Huang Thus the first objective of the present study was to and Hembree, 1979; Griswold et al., 1989). determine at the electron microscopic level using lanIt has been suggested that the regression of the ger- thanum nitrate as a tracer whether or not the interminal cells lodged in the adluminal compartment (i.e., Sertoli cell tight junctions broke down in VAD rats. above the blood-testis barrier) of the seminiferous epi- The second objective was to characterize the mechathelium in VAD rats was due to a breakdown of the inter-Sertoli tight junctions (Unni et al., 1983; Huang et al., 1988). Indeed Huang et al. (1988) reported that lanthanum nitrate, added to the intravascularly inReceived May 10, 1990; accepted in revised form June 19, 1990. jected glutaraldehyde fixative reached the lumen of the Address reprint requests to Dr. Carlos Morales, Department of Anatomy, seminiferous tubules due to the opening of inter-Ser- McGill University, 3640 University Street, Montreal, Quebec, Canada H3A 282. ~~~
0 1992 WILEY-LISS, INC.
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N. ISMAIL AND C.R. MORALES
nism of regression which resulted in the depletion of most germ cells from the seminiferous tubules of VAD rats. MATERIALS AND METHODS Four groups of male Sprague-Dawley rats (20 day old) were fed a viamin A deficient diet (U.S. Biochemical Corporation) for a different period of time (7,8,10, 12 weeks). Three rats were utilized for each group. The testes were fixed by perfusion via the abdominal aorta. The initial fixative used for perfusion was composed of 2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate with 0.1% calcium chloride (pH 7.6). Following perfusion the testes were removed, cut into small blocks (lmm3), and placed in the same fixative for 2 hours. The tissue was washed overnight in 0.1 M sodium cacodylate buffer. After overnight incubation, the tissue was impregnated in 1.5%potassium ferrocyanide reduced osmium (Karnovsky, 1971) for 1-2 hours. The tissue was then dehydrated in ascending concentrations of ethanol. In order t o use lanthanum as tracer, a modification of the procedure of Dym and Fawcett (1970) was used. A 2% lanthanum nitrate (Sigma Chemical Co.) solution was very slowly adjusted to pH 7.6-7.8 using 0.1 N NaOH. A barely visible precipitate was observed at this pH. The lanthanum nitrate thus prepared was then added to an equal volume of sodium cacodylate buffered glutaraldehyde. The final concentration of glutaraldehyde was 2.5%.Approximately 200 ml of the fixative mixture was perfused through the abdominal aorta over a period of 20 minutes. The tissue was then processed in a manner similar to that employed for the testes not treated with lanthanum except that 2% lanthanum nitrate was added to all solutions up to dehydration. The blocks of tissue were then embedded in Epon. Semithin sections (1 km thick) stained with toludine blue were prepared. Thin sections (exhibiting silvergold) of selected areas were cut with a diamond knife. The sections obtained from the lanthanum experiments were not stained, while the others were stained with uranyl acetate and lead citrate. The grids were observed on a Philips 301 Electron Microscope. RESULTS Status of Sertoli Cells Tight Junctions in VAD Rats In VAD rats the characteristic “ectoplasmic specializations” were always observed facing the latero-basal plasma membranes of Sertoli cells. These consisted of two adjacent Sertoli cell plasma membranes, flanked by two layers of cytoplasm containing bundles of filaments (actin) delimited by flattened cisternae of the endoplasmic reticulum (Fig. l).Facing such ectoplasmic specialization, the Sertoli cells of adjacent plasma membranes showed several focal points of close contact with a consequent occlusion of the intercellular space (Fig. 1).These focal sites of close apposition of plasma membranes correspond to sections through the linear tight junctions seen a t the interface of Sertoli cells and which constitute the blood-testis barrier (Dym and Fawcett, 1970; Russell, 1979).
In sections of seminiferous tubules of VAD rats perfused with a fixative containing the electron opaque lanthanum nitrate the tracer was readily detected in the interstitial space, on both sides of the myoid cells, and in the intercellular spaces of the basal compartment of the seminiferous epithelium (Fig. 2). Thus the tracer was seen in the thin intercellular spaces between spermatogonia and Sertoli cells or between adjacent Sertoli cells at the level of the basal compartment but was never seen in the adluminal compartment (Fig. 3, 4). The lanthanum was found to reach the inter-Sertoli cell tight junctions above the spermatogonial layer but was stopped a t that point (Figs. 3, 4). Morphological Analysis of Germ Cell Degeneration Seminiferous epithelium of 7- and 8-week VAD rats was lined by Sertoli cells, few type A spermatogonia, preleptotene spermatocytes (Pl), and few spermatids. Degenerating germ cells were frequently observed in the seminiferous epithelium of these animals. In toludine blue stained semithin sections examined with the light microscope, the degenerating germ cells were identified by their pyknotic nuclei. Degenerating spermatogonia were seen next to the basement membrane of the tubules, whereas degenerating spermatocytes were seen in the adluminal compartment. In this compartment large spherical multinucleated bodies were also seen. The acrosomic system seen at the surface of these nuclei permitted the identification of these bodies as fused spermatids (Fig. 5). Elongated spermatids (steps 10-15 approximately) were rarely observed but a few late spermatids (steps 16-19) were retained in the seminiferous epithelium. At the EM level, degenerating germ cells were identified by their increased nuclear and cytoplasmic density accompanied by a distortion of the nucleus and cytoplasmic organelles (Fig. 7). Seminiferous tubules of advanced stages of vitamin A deficiency (10-12 weeks) contained only Sertoli cells, few type A, spermatogonia, and occasional P1 spermatocytes (Fig. 6). Determination of Germ Cell Fate The cytoplasm of Sertoli cells of VAD rats contained numerous membrane bound phagosomes and phagosomes belonging to degenerating germ cells (Fig. 7). These structures were partly surrounded by Sertoli cell processes or completely embedded within the cytoplasm of Sertoli cells. Such phagosomes contained large membrane-delimited vacuoles, irregular membrane-delimited bodies with homogeneous or heterogeneous content of variable electron density, clusters of ribosomes, and condensed mitochondria (Fig. 7). Phagosomes of advanced degenerating cells showed severe distortion of the cytoplasmic organelles (Fig. 8). Such phagosomes also contained multilayered membranous profiles or electron dense bodies (Fig. 8). The Sertoli cell cytoplasm was also vacuolated and contained lipid droplets.
EFFECT OF VAD ON INTER-SERTOLI TIGHT JUNCTIONS
45
Fig. 1. Electron micrograph of a n ordinarily stained preparation of a Sertoli-Sertoli junction from a 10-week VAD rat perfused with 2.5%glutaraldehyde. The arrows indicate the focal site of tight junctions. Subsurface cisternae of endoplasmic reticulum were also present and separated from the cell surface by bundles of filaments. m, mitochondria; sp, spermatocyte. x 45,000.
DISCUSSION VAD Deficiency and the Inter-Sertoli Cell Tight Junctions The present investigation revealed that in VAD rats, the inter-Sertoli cell tight junctions remained intact even during the severe regression observed during weeks 8-11. At week 12 after the initiation of the feeding with the vitamin A deficient diet, the seminiferous epithelium was almost depleted of germ cells, and the Sertoli cell tight junctions were still effective in preventing the lanthanum nitrate from entering the adluminal compartment. This finding was in contradiction to the observations made by Huang et al. (1988), who reported that in VAD rats lanthanum was seen in the lumen of the seminiferous tubules within the intercellular spaces in the adluminal compartment or even within the cytoplasm of some Sertoli cells. In other studies where the testis showed severe degeneration and regression due to the suppression of circulating gonadotropins (%tale et al., 1973) or cryptorchidism (Stewart et al., 1988) the blood testis barrier remained intact.
Thus, the degeneration of germ cells occurring in VAD rats in not due to a breakdown of the inter-Sertoli cell tight junctions but confirms the notion that vitamin A has an essential role in the maintenance of germ cell proliferation and differentiation. The exact mechanism by which vitamin A acts on the process of spermatogenesis is still unknown, but recently a general scheme for the mode of action of retinol in the testis was proposed (Griswold et al., 1989).This scheme suggests that retinol is transported in the plasma to target tissue by retinol binding protein (RBP),which is normally complexed with transthyretin (TTR). Upon uptake of retinol by the cell, this substance forms a complex with cellular retinol binding protein (CRBP) followed perhaps by conversion to a retinyl ester that on hydrolysis would release retinol again. Retinoic acid can be formed from retinol, complexed with cellular retinoic acid binding protein (CRABP),and transferred to the retinoic acid receptor proteins. The receptorretinoic acid complex would then activate specific genes in target tissues. This scheme is speculative but several investigations have shown that Sertoli cells are
46
N.ISMAIL AND C.R. MORALES
Fig. 2. Electron micrograph of a testicular section from a 12-week VAD rat perfused with 2.5% glutaraldehyde containing 2% lanthanum nitrate. The electron opaque lanthanum has gained access to the base of the epithelium and had entered the intercellular space around a preleptotene sperrnatocyte (PL). At the top of the cell it can be seen that further penetration of the tracer has been prevented by intercellular tight junctions (arrows). my, myoid cell; S, Sertoli cell nucleus. x 14,000.
rich in CRBP but contain only low levels of CRABP, whereas the spermatocytes and spermatids contain high levels of CRABP (Huggenvik and Griswold, 1981; Porter et al., 1985; Blaner et al., 1987). Thus, according t o this model, transport of retinol to germinal cells in the adluminal compartment (uptake, esterification, and release in the adluminal space) must be mediated by Sertoli cells. A feature of this model involves the integrity of the Sertoli cell tight junctions. In consequence, if retinol is destined to the germinal cells, it must be transferred via the Sertoli cells. Non-esterified retinol, or retinoic acid which cannot be esterified, is rapidly metabolized and cannot be passed on the germinal cells. The fact that retinol but not retinoic acid reinitiates spermatogenesis (Howell et al., 1963; Ahluwalia and Bieri, 1971; Huang and Hembree, 1979)
also correlates well with the findings obtained in the present investigation.
Mode of Regression of the Seminiferous Epithelium in VAD Rats When vitamin A deficiency is started in the 20-dayold rats, the effect of the vitamin depletion takes several (approximately 7 to 8) weeks to manifest itself both on the body weight and on the testicular histology (Huang and Hembree, 1979; Huang and Marshall, 1983; Morales and Griswold, 1987; Ismail et al., 1990). During this period the seminiferous epithelium develops normally such that until 40-45 days spermatogenesis or the histological characteristics of the seminiferous epithelium of the rats fed with VAD diet does not
EFFECT OF VAD ON INTER-SERTOLI TIGHT JUNCTIONS
Fig. 3. Electron micrograph of a seminiferous epithelium of a 12week VAD rat showing lanthanum outlining 2 germ cells in the basal compartment. The tracer permeated the intercellular space of the basal compartment and was arrested by intact inter-Sertoli tight junctions (arrows). The intercellular space of the adluminal compartment was free of the tracer. my, myoid cell. X 5,000.
47
Fig. 4. Electron micrograph of seminiferous epithelium of a 12week VAD rat showing lanthanum outlining a spermatogenic cell in the basal compartment. The tracer is restricted to the basal compartment and prevented from permeation through the adluminal compartment by inter-Sertoli tight junctions (arrow). my, myoid cell; S, Sertoli cell nucleus. x 7,000.
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N. ISMAIL AND C.R. MORALES
Fig. 5. Light micrograph of a portion of a seminiferous tubule from a 7-week VAD rat. The seminiferous epithelium is lined with Setoli cell (S), preleptotene spermatocytes (PL), and few type A spermatogonia (A). Multinucleated degenerating round spermatids (arrowheads) are trapped in the apical cytoplasm of Sertoli cell. x 900.
Fig. 6. Light micrograph of seminiferous epithelium from advanced stage of vitamin A deficiency. The epithelium is lined by a single layer of Sertoli cell ( S ) and few type A spermatogonia (A). Degenerating germ cells (arrow) are enclosed within the apical cytoplasm of Sertoli cell. x 900.
differ from that of control rats. As the vitamin A deficiency becomes effective at the level of the testicular tissues, then it appears that all germinal cells are affected and the large majority of them do degenerate as already noted by various authors (Huang and Hembree, 1979; Huang and Marshall, 1983; Ismail et al., 1990) and verified in the present study. Degenerating germ cells are then phagocytosed by Sertoli cells and eliminated from the seminiferous epithelium. Only some type A spermatogonia (Ao, A,) and some preleptotene spermatocytes both arrested in their G2 phase (Ismail et al., 1990) are momentarily preserved. Thus, the regression of the epithelium is the consequence of the degeneration of germinal cells a t all steps of spermatogenesis. However a germ cell maturation deple-
tion process simultaneously takes place as the differentiating spermatogonia (i.e., the type A,, A,, A,, In, and B spermatogonia) degenerate resulting in the arrest of the production of more advanced germinal cells (spermatocytes and/or spermatids). Since the only germ cells present in the tubules of VAD rats for a period of time are type A, spermatogonia and preleptotene spermatocytes at the G2 phase of their respective cell cycle, when the animals are returned to a normal diet the epithelium is restored in a stage-synchronized manner as a result of the proliferation, differentiation, and renewals of these type A, spermatogonia as already described in previous studies (Morales and Griswold, 1987; Griswold et al., 1989; Ismail et al., 1990).
EFFECT O F VAD ON INTER-SERTOLI TIGHT JUNCTIONS
49
Fig. 7. Electron micrograph of Sertoli cell phagosome from a n 8week VAD rat containing a degenerating germ cell. The degenerating germ cell shows increased cytoplasmic density and contains organelles showing early signs of lysis. x 8,500.
Fig. 8. Electron micrograph of a 10-week VAD rat showing supranuclear cytoplasm of Sertoli cell. The cytoplasm contains three membrane-delimited phagosomes enclosing membranous structures and a heterogeneous content of variable electron density. Dense bodies characterized as lysosomes (L) are also present. These are membranedelimited elements with moderately electron dense matrix. x 18,000.
ACKNOWLEDGMENTS We thank Dr. Y. Clermont for valuable discussions and his help in the preparation of the manuscript. This work was supported by a grant from the Medical Research Council of Canada to Dr. C.R. Morales. Dr. Morales is fellow of the Fonds de la recherche en sante du Quebec (FRSQ).
A.V. (1988) Disruption of sustentacular (Sertoli) cell tight junctions and regression of spermatogenesis in vitamin A deficient rats. Acta Anat., 133:lO-15. Huggenvik, J., and Griswold, M.D. (1981). Retinol-binding protein in rat testicular cell. J. Reprod. Fert., 61:403-408. Ismail, N., Morales, C.R., and Clermond, Y. (1990) Role of spermatogonia in the stage-synchronization of the seminiferous epithelium in vitamin A deficient rats. Am. J . Anat., 188:57-63. Karnovsky, M.J. (1971) Use of ferrocyanide-reduced osmium tetroxCell Biol., Abstract ide in electron microscopy. Proc. llthAm. SOC. 284, p. 146. Morales, C.R., and Griswold, M.D. (1987) Retinol-induced stage synchronization in seminiferous tubules of the rat. Endocrinology, 121: 432-434 Porter, S.B., Ong, D.E., Chytil, F., and Orgebin-Crist, M.C. (1985) Localization of cellular retinol-binding protein and cellular retinoic acid-binding protein in the rat testis and epididymis. J . Androl., 6:197-212. Russell, L.D. (1979) Observations of the inter-relationships of Sertoli cells a t the level of the blood-testis barrier: Evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat. Am. J. Anat., 155:259-279. Stewart, R.J., Boyd, S., Brown, S., and Toner, P.G. (1990) The bloodtestis barrier in experimental unilateral cryptorchidism. J . Pathol., 1605-55. Unni, E., Rao, M.R.S., and Ganguly, J . (1983) Histological and ultrastructural studies on the effect of vitamin A depletion and subsequent repletion with vitamin A on germ cells and Sertoli cells in rat testis. Ind. J. Exp. Biol., 21:180-192. Vitale, R., Fawcett, D.W., and Dym, M. (1973) The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment. Anat. Rec., 176:333-344.
REFERENCES Ahluwalia, B., and Bieri, J.G. (1971) Local stimulatory effect of vitamin A on spermatogenesis in the rat. J. Nutr., 101:141-152. Blaner, W.S., Galdieri, M., and Goodman, D.S. (1987) Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells. Biol. Reprod., 36:130-137. Dym, M., and Fawcett, D.W. (1970) The blood testis barrier in the rat and the physiological compartmentation of the seminiferous tubules. Biol. Reprod., 3:308-326. Griswold, M.D., Bishop, P.D., Kim, K.-H., Ren, P., Siiteri, J.E., and Morales, C.R. (1989) The function of vitamin A in normal and synchronized seminiferous tubules. Ann. N.Y. Acad. Sci., 564:154-172. Howell, J. McC., Thompson, J.N., and Pitt, G.A. (1963) Histology of the lesions produced in the reproductive tract of animals fed a diet deficient in vitamin A alcohol but containing vitamin A acid. I. The male rat. J . Reprod. Fert., 5159-167. Huang, H.F.S., and Hembree, W.C. (1979) Spermatogenic response to vitamin A in vitamin A deficient rats. Biol. Reprod., 21:891-904. Huang, H.F.S., and Marshall, G.R. (1983) Failure of spermatid release under various vitamin A states-An indication of delayed spermiation. Biol. Reprod., 28:1163-1172. Huang, H.F.S., Yang, C.S., Meyenhofer, M., Gould, S., and Boccabella,
Effects of Vitamin A Deficiency on the Inter-Sertoli Cell Tight Junctions and on the Germ Cell Population NERMINE ISMAIL AND CARLOS R. MORALES Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2
KEY WORDS
Vitamin A deficiency, Blood-testis barrier, Seminiferous epithelium
ABSTRACT When 20-day-old rats are placed on a vitamin A deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells, a few residual AO,A, spermatogonia, and preleptotene spermatocytes (PL). The type A, spermatogonia and PL spermatocytes are arrested in their G, phase. In VAD rats type A,-A,, intermediate (In) and B spermatogonia and all types of spermatocytes (except PL spermatocytes) and spermatids are eliminated from the seminiferous tubules. Two questions were raised in this investigation: 1)Is there, in VAD rats, any correlation between a breakdown of the blood-testis barrier (e.g., Sertoli cell tight junctions) and germ cell loss? 2) Is the disappearance of most germinal cells due to their degeneration during spermatogenesis or to a maturation depletion process resulting from an arrest of spermatogenesis at the spermatogonial stage? To investigate these questions four groups of male SpragueDawley rats (20-days old) were fed a VAD diet for 7 to 12 weeks. The testes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M sodium cacodylate containing 2% lanthanum nitrate, an electron opaque tracer used to test the patency of Sertoli cell tight junctions. The lanthanum permeated the intercellular space of the basal compartment but was arrested by normal inter-Sertoli cell tight junctions. The seminiferous epithelium showed numerous degenerating germ cells, some being internalized by Sertoli cells as membrane-bound phagosomes. Thus, these results indicate firstly that inter-Sertoli cell tight junctions remain intact during vitamin A deficiency, and secondly that in a first phase nonviable germinal cells degenerate during spermatogenesis and their residues are actively phagocytosed by Sertoli cells followed by a second phase where the regressed state of the seminiferous epithelium is maintained by a maturation depletion condition resulting from an arrest of spermatogonial proliferation and differentation.
INTRODUCTION
toli cell tight junctions forming the blood-testis barrier. As a consequence, the modified milieu of the adluminal When 20-day-old rats are placed on a vitamin A de- compartment would not permit the normal differentificient diet (VAD) for a period of 12 weeks, the semi- ation of the spermatocytes and spermatids that would niferous tubules are found to contain only Sertoli cells, then degenerate. a few type A spermatogonia, and preleptotene sperAnother question raised by the regression of the sematocytes (Huang and Hembree, 1979; Morales and miniferous tubules in VAD rats was whether the deGriswold, 1987; Ismail et al., 1990). The type A sper- pletion of the germ cells from the epithelium was the matogonia, of the type A, class, are seemingly arrested consequence of the degeneration of the germinal cells in their G2phase of the cell cycle, whereas type A2-A4, that were in the process of differentiating into spermaintermediate, and type B spermatogonia and all types tozoa or of a maturation depletion process resulting of spermatocytes except a few preleptotene spermato- from the arrest of the proliferation of spermatogonia cytes are eliminated from the seminiferous epithelium with the consequence of a progressive disappearance (Ismail et al., 1990). Following this regression, sper- with time of the more advanced germinal cells from the matogenesis can be reinitiated by the administration of seminiferous epithelium or even of a combination of retinol but not by its metabolic derivative, retinoic acid these two processess. (Howell et al., 1963; Ahluwalia and Bieri, 1971; Huang Thus the first objective of the present study was to and Hembree, 1979; Griswold et al., 1989). determine at the electron microscopic level using lanIt has been suggested that the regression of the ger- thanum nitrate as a tracer whether or not the interminal cells lodged in the adluminal compartment (i.e., Sertoli cell tight junctions broke down in VAD rats. above the blood-testis barrier) of the seminiferous epi- The second objective was to characterize the mechathelium in VAD rats was due to a breakdown of the inter-Sertoli tight junctions (Unni et al., 1983; Huang et al., 1988). Indeed Huang et al. (1988) reported that lanthanum nitrate, added to the intravascularly inReceived May 10, 1990; accepted in revised form June 19, 1990. jected glutaraldehyde fixative reached the lumen of the Address reprint requests to Dr. Carlos Morales, Department of Anatomy, seminiferous tubules due to the opening of inter-Ser- McGill University, 3640 University Street, Montreal, Quebec, Canada H3A 282. ~~~
0 1992 WILEY-LISS, INC.
44
N. ISMAIL AND C.R. MORALES
nism of regression which resulted in the depletion of most germ cells from the seminiferous tubules of VAD rats. MATERIALS AND METHODS Four groups of male Sprague-Dawley rats (20 day old) were fed a viamin A deficient diet (U.S. Biochemical Corporation) for a different period of time (7,8,10, 12 weeks). Three rats were utilized for each group. The testes were fixed by perfusion via the abdominal aorta. The initial fixative used for perfusion was composed of 2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate with 0.1% calcium chloride (pH 7.6). Following perfusion the testes were removed, cut into small blocks (lmm3), and placed in the same fixative for 2 hours. The tissue was washed overnight in 0.1 M sodium cacodylate buffer. After overnight incubation, the tissue was impregnated in 1.5%potassium ferrocyanide reduced osmium (Karnovsky, 1971) for 1-2 hours. The tissue was then dehydrated in ascending concentrations of ethanol. In order t o use lanthanum as tracer, a modification of the procedure of Dym and Fawcett (1970) was used. A 2% lanthanum nitrate (Sigma Chemical Co.) solution was very slowly adjusted to pH 7.6-7.8 using 0.1 N NaOH. A barely visible precipitate was observed at this pH. The lanthanum nitrate thus prepared was then added to an equal volume of sodium cacodylate buffered glutaraldehyde. The final concentration of glutaraldehyde was 2.5%.Approximately 200 ml of the fixative mixture was perfused through the abdominal aorta over a period of 20 minutes. The tissue was then processed in a manner similar to that employed for the testes not treated with lanthanum except that 2% lanthanum nitrate was added to all solutions up to dehydration. The blocks of tissue were then embedded in Epon. Semithin sections (1 km thick) stained with toludine blue were prepared. Thin sections (exhibiting silvergold) of selected areas were cut with a diamond knife. The sections obtained from the lanthanum experiments were not stained, while the others were stained with uranyl acetate and lead citrate. The grids were observed on a Philips 301 Electron Microscope. RESULTS Status of Sertoli Cells Tight Junctions in VAD Rats In VAD rats the characteristic “ectoplasmic specializations” were always observed facing the latero-basal plasma membranes of Sertoli cells. These consisted of two adjacent Sertoli cell plasma membranes, flanked by two layers of cytoplasm containing bundles of filaments (actin) delimited by flattened cisternae of the endoplasmic reticulum (Fig. l).Facing such ectoplasmic specialization, the Sertoli cells of adjacent plasma membranes showed several focal points of close contact with a consequent occlusion of the intercellular space (Fig. 1).These focal sites of close apposition of plasma membranes correspond to sections through the linear tight junctions seen a t the interface of Sertoli cells and which constitute the blood-testis barrier (Dym and Fawcett, 1970; Russell, 1979).
In sections of seminiferous tubules of VAD rats perfused with a fixative containing the electron opaque lanthanum nitrate the tracer was readily detected in the interstitial space, on both sides of the myoid cells, and in the intercellular spaces of the basal compartment of the seminiferous epithelium (Fig. 2). Thus the tracer was seen in the thin intercellular spaces between spermatogonia and Sertoli cells or between adjacent Sertoli cells at the level of the basal compartment but was never seen in the adluminal compartment (Fig. 3, 4). The lanthanum was found to reach the inter-Sertoli cell tight junctions above the spermatogonial layer but was stopped a t that point (Figs. 3, 4). Morphological Analysis of Germ Cell Degeneration Seminiferous epithelium of 7- and 8-week VAD rats was lined by Sertoli cells, few type A spermatogonia, preleptotene spermatocytes (Pl), and few spermatids. Degenerating germ cells were frequently observed in the seminiferous epithelium of these animals. In toludine blue stained semithin sections examined with the light microscope, the degenerating germ cells were identified by their pyknotic nuclei. Degenerating spermatogonia were seen next to the basement membrane of the tubules, whereas degenerating spermatocytes were seen in the adluminal compartment. In this compartment large spherical multinucleated bodies were also seen. The acrosomic system seen at the surface of these nuclei permitted the identification of these bodies as fused spermatids (Fig. 5). Elongated spermatids (steps 10-15 approximately) were rarely observed but a few late spermatids (steps 16-19) were retained in the seminiferous epithelium. At the EM level, degenerating germ cells were identified by their increased nuclear and cytoplasmic density accompanied by a distortion of the nucleus and cytoplasmic organelles (Fig. 7). Seminiferous tubules of advanced stages of vitamin A deficiency (10-12 weeks) contained only Sertoli cells, few type A, spermatogonia, and occasional P1 spermatocytes (Fig. 6). Determination of Germ Cell Fate The cytoplasm of Sertoli cells of VAD rats contained numerous membrane bound phagosomes and phagosomes belonging to degenerating germ cells (Fig. 7). These structures were partly surrounded by Sertoli cell processes or completely embedded within the cytoplasm of Sertoli cells. Such phagosomes contained large membrane-delimited vacuoles, irregular membrane-delimited bodies with homogeneous or heterogeneous content of variable electron density, clusters of ribosomes, and condensed mitochondria (Fig. 7). Phagosomes of advanced degenerating cells showed severe distortion of the cytoplasmic organelles (Fig. 8). Such phagosomes also contained multilayered membranous profiles or electron dense bodies (Fig. 8). The Sertoli cell cytoplasm was also vacuolated and contained lipid droplets.
EFFECT OF VAD ON INTER-SERTOLI TIGHT JUNCTIONS
45
Fig. 1. Electron micrograph of a n ordinarily stained preparation of a Sertoli-Sertoli junction from a 10-week VAD rat perfused with 2.5%glutaraldehyde. The arrows indicate the focal site of tight junctions. Subsurface cisternae of endoplasmic reticulum were also present and separated from the cell surface by bundles of filaments. m, mitochondria; sp, spermatocyte. x 45,000.
DISCUSSION VAD Deficiency and the Inter-Sertoli Cell Tight Junctions The present investigation revealed that in VAD rats, the inter-Sertoli cell tight junctions remained intact even during the severe regression observed during weeks 8-11. At week 12 after the initiation of the feeding with the vitamin A deficient diet, the seminiferous epithelium was almost depleted of germ cells, and the Sertoli cell tight junctions were still effective in preventing the lanthanum nitrate from entering the adluminal compartment. This finding was in contradiction to the observations made by Huang et al. (1988), who reported that in VAD rats lanthanum was seen in the lumen of the seminiferous tubules within the intercellular spaces in the adluminal compartment or even within the cytoplasm of some Sertoli cells. In other studies where the testis showed severe degeneration and regression due to the suppression of circulating gonadotropins (%tale et al., 1973) or cryptorchidism (Stewart et al., 1988) the blood testis barrier remained intact.
Thus, the degeneration of germ cells occurring in VAD rats in not due to a breakdown of the inter-Sertoli cell tight junctions but confirms the notion that vitamin A has an essential role in the maintenance of germ cell proliferation and differentiation. The exact mechanism by which vitamin A acts on the process of spermatogenesis is still unknown, but recently a general scheme for the mode of action of retinol in the testis was proposed (Griswold et al., 1989).This scheme suggests that retinol is transported in the plasma to target tissue by retinol binding protein (RBP),which is normally complexed with transthyretin (TTR). Upon uptake of retinol by the cell, this substance forms a complex with cellular retinol binding protein (CRBP) followed perhaps by conversion to a retinyl ester that on hydrolysis would release retinol again. Retinoic acid can be formed from retinol, complexed with cellular retinoic acid binding protein (CRABP),and transferred to the retinoic acid receptor proteins. The receptorretinoic acid complex would then activate specific genes in target tissues. This scheme is speculative but several investigations have shown that Sertoli cells are
46
N.ISMAIL AND C.R. MORALES
Fig. 2. Electron micrograph of a testicular section from a 12-week VAD rat perfused with 2.5% glutaraldehyde containing 2% lanthanum nitrate. The electron opaque lanthanum has gained access to the base of the epithelium and had entered the intercellular space around a preleptotene sperrnatocyte (PL). At the top of the cell it can be seen that further penetration of the tracer has been prevented by intercellular tight junctions (arrows). my, myoid cell; S, Sertoli cell nucleus. x 14,000.
rich in CRBP but contain only low levels of CRABP, whereas the spermatocytes and spermatids contain high levels of CRABP (Huggenvik and Griswold, 1981; Porter et al., 1985; Blaner et al., 1987). Thus, according t o this model, transport of retinol to germinal cells in the adluminal compartment (uptake, esterification, and release in the adluminal space) must be mediated by Sertoli cells. A feature of this model involves the integrity of the Sertoli cell tight junctions. In consequence, if retinol is destined to the germinal cells, it must be transferred via the Sertoli cells. Non-esterified retinol, or retinoic acid which cannot be esterified, is rapidly metabolized and cannot be passed on the germinal cells. The fact that retinol but not retinoic acid reinitiates spermatogenesis (Howell et al., 1963; Ahluwalia and Bieri, 1971; Huang and Hembree, 1979)
also correlates well with the findings obtained in the present investigation.
Mode of Regression of the Seminiferous Epithelium in VAD Rats When vitamin A deficiency is started in the 20-dayold rats, the effect of the vitamin depletion takes several (approximately 7 to 8) weeks to manifest itself both on the body weight and on the testicular histology (Huang and Hembree, 1979; Huang and Marshall, 1983; Morales and Griswold, 1987; Ismail et al., 1990). During this period the seminiferous epithelium develops normally such that until 40-45 days spermatogenesis or the histological characteristics of the seminiferous epithelium of the rats fed with VAD diet does not
EFFECT OF VAD ON INTER-SERTOLI TIGHT JUNCTIONS
Fig. 3. Electron micrograph of a seminiferous epithelium of a 12week VAD rat showing lanthanum outlining 2 germ cells in the basal compartment. The tracer permeated the intercellular space of the basal compartment and was arrested by intact inter-Sertoli tight junctions (arrows). The intercellular space of the adluminal compartment was free of the tracer. my, myoid cell. X 5,000.
47
Fig. 4. Electron micrograph of seminiferous epithelium of a 12week VAD rat showing lanthanum outlining a spermatogenic cell in the basal compartment. The tracer is restricted to the basal compartment and prevented from permeation through the adluminal compartment by inter-Sertoli tight junctions (arrow). my, myoid cell; S, Sertoli cell nucleus. x 7,000.
48
N. ISMAIL AND C.R. MORALES
Fig. 5. Light micrograph of a portion of a seminiferous tubule from a 7-week VAD rat. The seminiferous epithelium is lined with Setoli cell (S), preleptotene spermatocytes (PL), and few type A spermatogonia (A). Multinucleated degenerating round spermatids (arrowheads) are trapped in the apical cytoplasm of Sertoli cell. x 900.
Fig. 6. Light micrograph of seminiferous epithelium from advanced stage of vitamin A deficiency. The epithelium is lined by a single layer of Sertoli cell ( S ) and few type A spermatogonia (A). Degenerating germ cells (arrow) are enclosed within the apical cytoplasm of Sertoli cell. x 900.
differ from that of control rats. As the vitamin A deficiency becomes effective at the level of the testicular tissues, then it appears that all germinal cells are affected and the large majority of them do degenerate as already noted by various authors (Huang and Hembree, 1979; Huang and Marshall, 1983; Ismail et al., 1990) and verified in the present study. Degenerating germ cells are then phagocytosed by Sertoli cells and eliminated from the seminiferous epithelium. Only some type A spermatogonia (Ao, A,) and some preleptotene spermatocytes both arrested in their G2 phase (Ismail et al., 1990) are momentarily preserved. Thus, the regression of the epithelium is the consequence of the degeneration of germinal cells a t all steps of spermatogenesis. However a germ cell maturation deple-
tion process simultaneously takes place as the differentiating spermatogonia (i.e., the type A,, A,, A,, In, and B spermatogonia) degenerate resulting in the arrest of the production of more advanced germinal cells (spermatocytes and/or spermatids). Since the only germ cells present in the tubules of VAD rats for a period of time are type A, spermatogonia and preleptotene spermatocytes at the G2 phase of their respective cell cycle, when the animals are returned to a normal diet the epithelium is restored in a stage-synchronized manner as a result of the proliferation, differentiation, and renewals of these type A, spermatogonia as already described in previous studies (Morales and Griswold, 1987; Griswold et al., 1989; Ismail et al., 1990).
EFFECT O F VAD ON INTER-SERTOLI TIGHT JUNCTIONS
49
Fig. 7. Electron micrograph of Sertoli cell phagosome from a n 8week VAD rat containing a degenerating germ cell. The degenerating germ cell shows increased cytoplasmic density and contains organelles showing early signs of lysis. x 8,500.
Fig. 8. Electron micrograph of a 10-week VAD rat showing supranuclear cytoplasm of Sertoli cell. The cytoplasm contains three membrane-delimited phagosomes enclosing membranous structures and a heterogeneous content of variable electron density. Dense bodies characterized as lysosomes (L) are also present. These are membranedelimited elements with moderately electron dense matrix. x 18,000.
ACKNOWLEDGMENTS We thank Dr. Y. Clermont for valuable discussions and his help in the preparation of the manuscript. This work was supported by a grant from the Medical Research Council of Canada to Dr. C.R. Morales. Dr. Morales is fellow of the Fonds de la recherche en sante du Quebec (FRSQ).
A.V. (1988) Disruption of sustentacular (Sertoli) cell tight junctions and regression of spermatogenesis in vitamin A deficient rats. Acta Anat., 133:lO-15. Huggenvik, J., and Griswold, M.D. (1981). Retinol-binding protein in rat testicular cell. J. Reprod. Fert., 61:403-408. Ismail, N., Morales, C.R., and Clermond, Y. (1990) Role of spermatogonia in the stage-synchronization of the seminiferous epithelium in vitamin A deficient rats. Am. J . Anat., 188:57-63. Karnovsky, M.J. (1971) Use of ferrocyanide-reduced osmium tetroxCell Biol., Abstract ide in electron microscopy. Proc. llthAm. SOC. 284, p. 146. Morales, C.R., and Griswold, M.D. (1987) Retinol-induced stage synchronization in seminiferous tubules of the rat. Endocrinology, 121: 432-434 Porter, S.B., Ong, D.E., Chytil, F., and Orgebin-Crist, M.C. (1985) Localization of cellular retinol-binding protein and cellular retinoic acid-binding protein in the rat testis and epididymis. J . Androl., 6:197-212. Russell, L.D. (1979) Observations of the inter-relationships of Sertoli cells a t the level of the blood-testis barrier: Evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat. Am. J. Anat., 155:259-279. Stewart, R.J., Boyd, S., Brown, S., and Toner, P.G. (1990) The bloodtestis barrier in experimental unilateral cryptorchidism. J . Pathol., 1605-55. Unni, E., Rao, M.R.S., and Ganguly, J . (1983) Histological and ultrastructural studies on the effect of vitamin A depletion and subsequent repletion with vitamin A on germ cells and Sertoli cells in rat testis. Ind. J. Exp. Biol., 21:180-192. Vitale, R., Fawcett, D.W., and Dym, M. (1973) The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment. Anat. Rec., 176:333-344.
REFERENCES Ahluwalia, B., and Bieri, J.G. (1971) Local stimulatory effect of vitamin A on spermatogenesis in the rat. J. Nutr., 101:141-152. Blaner, W.S., Galdieri, M., and Goodman, D.S. (1987) Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells. Biol. Reprod., 36:130-137. Dym, M., and Fawcett, D.W. (1970) The blood testis barrier in the rat and the physiological compartmentation of the seminiferous tubules. Biol. Reprod., 3:308-326. Griswold, M.D., Bishop, P.D., Kim, K.-H., Ren, P., Siiteri, J.E., and Morales, C.R. (1989) The function of vitamin A in normal and synchronized seminiferous tubules. Ann. N.Y. Acad. Sci., 564:154-172. Howell, J. McC., Thompson, J.N., and Pitt, G.A. (1963) Histology of the lesions produced in the reproductive tract of animals fed a diet deficient in vitamin A alcohol but containing vitamin A acid. I. The male rat. J . Reprod. Fert., 5159-167. Huang, H.F.S., and Hembree, W.C. (1979) Spermatogenic response to vitamin A in vitamin A deficient rats. Biol. Reprod., 21:891-904. Huang, H.F.S., and Marshall, G.R. (1983) Failure of spermatid release under various vitamin A states-An indication of delayed spermiation. Biol. Reprod., 28:1163-1172. Huang, H.F.S., Yang, C.S., Meyenhofer, M., Gould, S., and Boccabella,
