JOURNAL OF ULTRASTRUCTURE RESEARCH 57, 5 4 - 6 4
(1976)
The Perinuclear Substance of Boar, Bull, Ram and Rabbit Spermatozoa J . L. COURTENS, 1 M. COUROT, 1 AND J. E. FLECHON ~
Station de Physiologie de la Reproduction, I.N.R.A., 37380 Nouzillyl; Station centrale de Physiologie anirnale, I.N.R.A., 78350 Jouy-en-Josas,2France Received February 10, 1976 The perinuclear substance (PNS) in boar, bull, ram and rabbit spermatozoa is described. The PNS is present as a thin, continuous layer of lysine-rich proteins located between the nucleus on one side and the acrosome and postacrosomal lamina on the other. The perforatorium and equatorial blisters (rabbit) are part of the PNS. PNS first appears when the acrosomal vesicle apposes on the nucleus of young spermatids. Morphological and cytochemical modifications of PNS occur during spermiogenesis and epididymal maturation. The functional role of this sperm cell entity is briefly discussed. L'~tude de la substance p~rinucldaire (SPN) est effectu~e dans les spermatozo~des de Verrat, Taureau, B~lier et Lapin. La SPN est constitute d'une fine couche de prot~ines riches en lysine, dispos~e de mani~re continue entre le noyau d'une part, l'acrosome et la lame postacrosomique d'autre part. Le perforatorium et les ampoules 6quatoriales (Lapin) font partie de la SPN. La SPN apparalt pour la premiere fois au moment de l'accolement de la v~sicule acrosomique sur le noyau des jeunes spermatides. Des modifications morphologiques et cytochimiques de la SPN se produisent au cours de la spermiogen~se et de la maturation ~pididymaire. Le rSle fonctionnel de cette entit~ du gamete m~le est discut~. ( P N S ) for al l t h e m a t e r i a l l o c a t e d b e t w e e n t h e n u c l e a r e n v e l o p e a n d t h e a c r o s o m e or p o s t a c r o s o m a l l a m i n a (3, 12, 23). Is t h e P N S a s t r u c t u r a l , b i o c h e m i c a l ent i t y ? T h e p u r p o s e of t h i s w o r k is to des c r i b e t h e d i s t r i b u t i o n of P N S , its o r i g i n a n d c y t o c h e m i c a l c o m p o s i t i o n i n t h e sp er m a t o z o a of c o m m o n f a r m a n i m a l s .
M o s t of t h e t r a n s m i s s i o n e l e c t r o n m i c r o scope s t u d i e s on t h e h e a d of m a m m a l i a n s p e r m a t o z o a h a v e d e m o n s t r a t e d the prese n c e of m a t e r i a l l o c a t e d b e t w e e n t h e acros o m e a n d t h e n u c l e a r e n v e l o p e , i n t h e soca]led s u b a c r o s o m a l space. S o m e a u t h o r s h a v e n o t r e c o g n i z e d t h e e x i s t e n c e of t h i s m a t e r i a l (10), or a t l e a s t i ts s i g n i f i c a n c e (11). O n l y J o n e s (20, 21) d e s c r i b e d a conn e c t i o n b e t w e e n t h e p e r f o r a t o r i u m (or apical body), t h e s u b a c r o s o m a l s u b s t a n c e a n d t h e m a t e r i a l p r e s e n t on t h e n u c l e a r e n v e lope u n d e r t h e p o s t a c r o s o m a l l a m i n a in ram and boar spermatozoa. Continuous p e r i n u c l e a r m a t e r i a l was also i n c i d e n t a l l y s h o w n i n s p e r m of g o l d e n h a m s t e r , r a b b i t , a n d t u p a i a (1, 17, 19). We s h a l l r e s t r i c t t h e t e r m s ~¢perforator i u m " a n d " s u b a c r o s o m a l s u b s t a n c e " to t h e i r c l a s s i c a l m e a n i n g s (24), a n d u s e t h e general term "perinuclear substance"
MATERIAL AND METHODS
Morphological S t u d i e s Ram testis and rabbit and ram epididymis were fixed by perfusion or immersion with glutaraldehyde fixative. Various combinations of glutaraldehyde in phosphate or cacodylate buffers were used for fixation of ejaculated (and uterine) sperm of boar, bull, rabbit, and ram. The material was embedded either in glycol methacrylate or, after eventual osmium tetroxide prefixation, in (agar and) Epon, in a mixture of Araldite and Epon or in Spurr's medium (27). Fixed sperm cells were also processed by ultracryotomy (30). Ultrathin sections were stained either with alcoholic or aqueous uranyl acetate and lead citrate or in alcoholic solution of phosphotungstic acid.
1J.L.C. and M.C. 2 J.E.F. 54 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
Cytochemical Studies of Protein Amino Residues Ram testis was fixed by immersion in 4% glutaraldehyde in 0.1 M phosphate buffer. After inhibiting sulfhydryl and guanidyl groups with n-ethyl maleimide and malonaldehyde, respectively (1 and 2%, pH 7.4, 4 hr), the tissue was stained en bloc by alcoholic phosphotungstic acid (PTA/e) or hydroquinone-ferricyanide-copper phosphotungstic acid (HQ-Fe) to locate lysine residues according to Courtens and Loir (7). In some cases no inhibition was done before PTA/e treatment. Ultrathin sections in the Epon-embedded material were observed without any post-staining. RESULTS
Morphology of the Perinuclear Substance in Mature Spermatozoa In all types of fixations and embeddings used and after nonspecific staining, the spermatozoa show a continuous layer of p e r i n u c l e a r substance (PNS) u n d e r the acrosome and the postacrosomal lamina. The aspect of this substance differs according to the head region. The acrosomal region. In front of the a n t e r i o r nuclear edge, a large a m o u n t of the PNS is in the shape of a wedge, previously referred to as the p e r f o r a t o r i u m or apical body (Figs. 1 and 2); it appears to follow the horseshoe shape of the m a r g i n a l t h i c k e n i n g (Fig. 3). Towards the posterior end of the a n t e r i o r segment, where the m a r g i n a l t h i c k e n i n g decreases, the PNS also fills the p e r i n u c l e a r space over the edge of the nucleus, b u t does not r e t a i n its wedge shape in t r a n s v e r s e sections (Figs. 4-7, 9). On the dorsal and v e n t r a l sides of the nucleus, the P N S appears as a thin l a y e r in the e n t i r e subacrosomal space (Figs. 1, 2, 4-7, 9, 10); it is t h i c k e r u n d e r the equato• rial segment, especially at its limit with the a n t e r i o r segment (Figs. 8, 10, 13). In rabbit spermatozoa, the P N S is very expanded in this location, i.e., it forms at least two blisters of spongious, or sometimes homogeneous, m a t e r i a l on both sides of the nucleus (Figs. 6 and 9). The postacrosomal region. The PNS in
55
the postacrosomal region is clearly contiguous with t h a t in the subacrosomal space, and a small p a r t of it also appears to surround the posterior rim of the equatorial segment (Figs. 13, 14, 16). The PNS does not completely fill the space between the nucleus and the postacrosomal l a m i n a and appears loosely connected to the l a t t e r (Figs. 11-16). On sagittal sections, the posterior part of the PNS appears s t r a i g h t and closely applied on a portion of thickened nuclear envelope; it is h e r e a f t e r referred to as the ~postnuclear band" (Figs. 12, 14, 15). The ends of the PNS and postacrosomal l a m i n a are pinched t o g e t h e r u n d e r the posterior ring w h e r e the p l a s m a m e m b r a n e m a k e s a groove in the direction of the nuclear envelope (Figs. 11, 14, 15). M a t u r e rabbit spermatozoa show clear spaces between the nucleus and the PNS in the postacrosomal region (Fig. 15). However, these spaces are not present in i m m a t u r e and u t e r i n e spermatozoa (Figs. 14 and 16).
Origin and Chemical Composition of P N S of Ram Spermatozoa After HQ-Fe staining, the PNS is visible between nucleus and acrosome (Fig. 17) as soon as the acrosomal vesicle adheres to the nucleus (stages 1-3) (5). The PNS extends and its electron density increases (Fig. 18) as the acrosome cap spreads over the r o u n d spermatid nucleus (stages 4-8). The postnuclear band appears in the posterior part of the subacrosomal space during stages 9 and 10 and r e m a i n s in this location from stages 9 to 12. It consists of two components which join to form a diss y m m e t r i c a l V pointing to the anterior of the head (Figs. 19 and 20). The densely stained P N S extends into the postacrosomal region d u r i n g stage 12 concomitantly with the m i g r a t i o n of the postnuclear band and the perinuclear ring. The postnuclear band slips down between the nuclear envelope and the plasma membrane, during which time both its arms
56
COURTENS, COUROT AND FLECHON
Q P
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
become apposed on the nearest of these membranes (Fig. 21). The postnuclear band later remains as a clearly defined belt around the posterior end of the nucleus, in front of the posterior ring; the external arm of the V vanishes, and the internal arm is then anteriorly covered by the postacrosomal lamina applied along the plasma membrane (Fig. 22). At the end of stage 12 the perinuclear ring disappears. From stage 13 to spermiation, the postnuclear band becomes very narrow and entirely covered by the postacrosomal dense lamina (Fig. 12). When arginine is blocked with malonaldehyde, only the postnuclear band is stained by en bloc alcoholic PTA in spermatids (Fig. 20) and testicular spermatozoa, whereas all the PNS reacts in spermatoz from cauda epididymis. All the results obtained are summarized in Table I. DISCUSSION
Morphological Entity of PNS The PNS, visualized by various techniques including ultracryotomy (this study) and freeze-fracturing (13), constitutes a continuous layer under the acrosome and postacrosomal lamina in all the paddle-shaped spermatozoa studied. No wide species differences appear in the morphology of different PNS regions. For example, the PNS is thicker at the limit of the anterior and equatorial seg-
57
ments in the spermatozoa of the four species. However, the external development of this part of the PNS into blisters [from the mature spermatid stage (25)] is unique to rabbit and hare (24). The fact that the PNS is an entity is also shown by its genesis. PNS is laid down in ram spermatids as soon as a subacrosomal space exists, and also in rat (22) and golden hamster (16, 18). During elongation and flattening of the spermatid nucleus, PNS accumulates all around the edge of the nucleus under the acrosome. It assumes a wedge shape (the perforatorium) only under the anterior part of the marginal thickening, after elongation of the corresponding subacrosomal space [before spermiation in rabbit (25)]. In the postacrosomal region, the PNS appears to be an extension of the already present subacrosomal substance, consecutive to the migration of the postnuclear band, first described as "anneau postnucl~aire" (7). To avoid confusion with the posterior ring, the term postnuclear band is to be added to the sperm nomenclature proposed by some of us (3). It does not seem important whether the postnuclear band is a part of the PNS or only its posterior boundary. Although appearing at the same time in ram [as in mouse (26)], the highly differentiated postacrosomal lamina (14) cannot be confused with the underlying PNS.
Fia. 1. Sagittal section in the anterior part of a bull sperm head from cauda epididymis. The PNS is continuously spread between the nucleus and the acrosome and forms a finger-like anterior projection, the perforatorium (P). Glut, E, U and Pb; × 100 000. Abbreviations for all figures: Sperm morphology: A: acrosome; AP: arrays of particles; AV: acrosomal vesicle; AS: anterior segment of the acrosome; ES: equatorial segment of the acrosome; CC: crystalline core (in marginal thickening); DC: dense core (in m a r g i n a l thickening); G: acrosomal granule; N: nucleus; NE: nuclear envelope; P: perforatorium; PAL: postacrosomal lamina; PNB: postnuclear band; PR: posterior ring. Techniques: PTA/e: phosphotungstic acid in ethanol; E: Epon; Glut: glutaraldehyde; GMA: glycol methacrylate; HQ-Fe: hydroquinone-ferricyanidecopper phosphotungstic acid; Pb: lead citrate; U: uranyl acetate. Fia. 2. Similar section of an ejaculated rabbit spermatozoon. The PNS, including the wedge-like perfora~ torium (P), is visible between the acrosome and the nuclear envelope (NE). Glut, agar E, U and Pb; × 100 000. Fro. 3. Anterior portion of a n ejaculated r a m spermatozoon sectioned approximately parallel to the flat surface of the nucleus (N). The perforatorium is situated between the nucleus and the tip of the acrosome containing paracrystalline m a t e r i a l ( ~" ~ ). Glut, E, U and Pb; × 60 000.
58
COURTENS, COUROT AND FLt~CHON
Nature and Uniformity of PNS Chemical Composition Starting from its deposition, the PNS is a proteinaceous material, as first suggested by Nicander and Bane (24); it is apparently devoid of sugar residues since we failed to stain it with the techniques of Thi~ry and Rambourg (29). As shown by HQ-Fe staining after use of inhibitors, lysine residues seem to gather in the acrosomal and forming postacrosomal regions of the PNS at the same time as lysine-rich histones leave the nucleus in a
wave, progressing towards its posterior end (7). The disappearance of nuclear pores, as described in mouse (26), is probably related to the chromatin condensation and the changes in the PNS. Alcoholic PTA stainability after malonaldehyde inhibition increases during epididymal maturation in the whole PNS (except in the already stained postnuclear band). This may indicate a reduction in the distance between protein lysine groups (7). In fact, a condensation in the PNS and a shortening of the perforatorium were described at the same time in boar spermato-
FIG. 4. Transverse section of an ejaculated bull sperm head in the caudal part of the anterior segment. The PNS is thin, except on the lateral edges of the nucleus where it occupies the free space under the marginal thickening. Glut, E, U and Pb; × 50 000. FIG. 5. Slightly oblique section of a rabbit sperm head in the anterior segment. The continuous PNS shows a finger-like tip, the perforatorium (P) on the more anterior side (larger marginal thickening) and a blunt extremity in a posterior location (opposite end of the section). Glut, agar E, U and Pb; × 45 000. FIG. 6. Transverse section of a rabbit sperm head in the posterior part of the anterior segment. The PNS is thick on the lateral edges and developed in two spongious blisters (B) on t h e ventral and dorsal sides. Glut, agar E, U and Pb; x 56 000. FIG. 7. Oblique section in the posterior extremity of the anterior segment and in the equatorial segment of a rabbit sperm head. The PNS is thin under the equatorial segment (ES), except on the lateral edges and especially at the contact with the anterior segment. Glut, agar E, U and Pb; × 45 000. FIG. 8. Sagittal section of'a boar sperm head at the limit of the two acrosomal segments (AS and ES). The PNS fills the subacrosomal space, larger in this location. Glut, ultrathin frozen section, U and Pb; x 96 000. FIG. 9. Transverse sections of a rabbit sperm head in the region of the blisters (B) whicl~/extend over the lateral ridge (B'). Between the blisters and on the other lateral ridge (top) the PNS is thick. Acrosome and PNS are stained, as the technique used may be specific for proteins. Glut, GMA, PTA/e on the thin section; × 60 000. FIG. 10. Sagittal sections of boar sperm heads. The PNS is continuous in the subacrosomal space and thicker at the limit of the two acrosomal segments (--*). Glut, agar E, PTA/e on the thin section; x 40 000. FIG. 11. Postacrosomal region of a boar sperm head. The different stained layers from outside to inside are: plasma membrane, postacrosoma] lamina, PNS, nuclear envelope. As in the preceding picture, the staining used on sections has no known specificity, but outlines the PNS very well. Glut, .agar E, U in methanol and Pb; x 50 000. Fro. 12. Sagittal section of a ram testicular sperm head. The PNS appears to end posteriorly in a dense layer or postnuclear band (PNB) located between the postacrosomal lamina (PAL) and the nuclear envelope (NE). Glut, n-ethyl maleimide + malonaldehyde, HQ-Fe, E; x 100 000. FIG. 13. Sagitta] section of a bull sperm head from cauda epididymis: the PNS is contiguous under the acrosome and postacrosomal lamina (PAL); it is thicker at the limit of the equatorial segments (AS and ES) and surrounds the posterior end of the ES. Glut, E, U and Pb; × 60 000. FIG. 14. Sagittal section of a rabbit sperm head from corpus epididymis: contiguity of the PNS under the equatorial segment (ES) and the postacrosomal lamina (PAL). The PNS is caudally ended by the postnuclear band (PNB) inserted between the nuclear envelope (NE) and a thickened portion of the postacrosomal lamina, in front of the posterior ring (PR). Glut, E, U and Pb; × 65 000. FIG. 15. Similar section of an ejaculated rabbit sperm; a clear space (~-) is visible between the nucleus and the PNS under the postacrosomal lamina (PAL), except in the anterior and posterior extremities, The postnuclear band (PNB) is still applied on the nuclear envelope. Glut, agar E, U and Pb; x 84 000. FIG. 16. Rabbit spermatozoon recovered in the uterus 12 hr after coitus. The morphology is the same as for immature sperm. The postnuclear band is not visible in this slightly oblique section. Glut, E, U and Pb; × 64 000.
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
59
• PNS
PNS
Q
Q
AS
PNS
(9
60
COURTENS, COUROT AND FL~CHON
B'
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
~®
61
®
62
COURTENS, COUROT AND FL~CHON
63
PERINUCLEAR SUBSTANCE OF SPERMATOZOA TABLE I CYTOCHEMISTRY OF PNS = PTA/e
PTA/e (after malonaldehyde blockade)
= HQ-Fe
Testis
+
-
+
Cauda epididymis
+
+
+
Groups of 7-8. Lys in prox.
Lysine
Ejaculated spermatozoa
+ (on thin sections in GMA)
Nature of constituents
Arginine and lysine
zoa (21). Finally, disulfide bonds, already present in the spermatid PNS (6), also increase during epididymal maturation (4). Such crosslinked proteins are relatively resistant to pronase extraction on thin sections of rabbit spermatozoa (15).
Role of the PNS The fact that the PNS appears as a structural and biochemical entity also suggests t h a t it plays a functional role. This role may be important for fertilization, as the PNS is the underlying substrate of the acrosome and postacrosomal lamina, the sperm elements which are related to egg penetration and attachment. As pointed out previously (9, 12, 16, 23), the PNS may act as a cement between the nucleus and the overlaying elements of the sperm head. In the postacrosomal region, the postacrosomal lamina and PNS are spatially linked, since in m a t ur e rabbit spermatozoa their mutual opposition is
not d i s t u r b e d by the presence of clear spaces over the nucleus. These spaces, reported to contain phospholipid material (28), disappear in uterine spermatozoa and the PNS returns close to the nuclear surface. In the acrosomal region the cement hypothesis is substantiated by the persistence of the inner acrosomal membrane on spermatozoa deprived of the anterior segment (2) and in the process of ovum penetration (7, 17, 31). The scleroprotein nat ure of the PNS may also give rigidity to the perforatorium, thus facilitating penetration of the zona pellucida (31). The technical assistance of D. Huneau is gratefully acknowledged. REFERENCES 1. BEDFORD,J. M., Contribution Primatology 3, 97 (1974). 2. BROWN, C. R., ANDANI, Z., AND HARTREE, E. F.,
FIG. 17-22. Spermiogenesis in the ram (glut, n-ethyl maleimide + malonaldehyde, HQ-Fe, E, except Fig. 21: PTA/e instead of HQ-Fe). FIG. 17. At stage 3, the stained PNS is visible between the dense peripheral chromatin and the membrane ( $ ) of the acrosomal vesicle (AV). × 53 000. FIG. 18. At stage 6, a thicker layer of PNS is interposed between the nucleus and the acrosomal vesicle containing an acrosomal granule (G). × 38 000. Fro. 19. Elongated spermatid (stage 11); the PNS is about as thick and stained as the acrosome (A). In front of the perinuclear ring (--~), both show a thickened end, V-shaped in the case of the PNS: the future postnuclear band (PNB). x 43 000. Fia. 20. The postnuclear band (PNB) appears as V-shaped stained material located in front of the perinuclear ring (-~), and between the posterior extremity of the acrosome (A) and a thickened portion of nuclear envelope (NE). In front of the postnuclear band, the PNS is not stained (stage 10). x 43 000. FIG. 21. During the sliding of the perinuclear ring (--~), the postnuclear band (PNB) follows the former. x 34 000. Fro. 22. At the end of stage 12, the perinuclear ring (-->) and postnuclear band (PNB) have reached the posterior part of the nucleus. PNS has deposited under the postacrosomal lamina (PAL). × 35 000.
64
COURTENS, COUROT AND FL~CHON
Biochem. J. 149, 133 (1975). 3. BUSTOS-OBREGON,E., COUROT, M., FL~CHON, J. E., HOCHEREAUDE REVIERS, M. J., AND HOLSTEIN, A. F., Andrologia 7, 141 (1975). 4. CALVIN, H. I., AND BEDFORD, J. M., J. Reprod. Fertil. Suppl. 13, 65 (1971). 5. CLERMONT, Y., AND LEBLOND, C. P., Arner. J. Anat. 96, 229 (1955). 6. COURTENS,J. L., unpublished data (1975). 7. COURTENS,J. L., AND LOIR, M., J. Microsc. Biol. Cell. 24, 249 (1975). 8. COURT~NS,J. L., AND LOIR, M., J. Microsc. Biol. Cell. 24, 259 (1975). 9. FAWCETT, D. W., Biol, Reprod. Suppl. 2, 90 (1970). 10. FAWC~TT, D. W., AND ITO, S., Amer. J. Anat. 116, 567 (1965). 11. FAWCETT, D. W., AND PHILLIPS, D. M., J. Reprod. Fertil. Suppl. 6, 405 (1969). 12. FLI~CHON, J. E., thesis, University of Paris, 1973. 13. FLI~CHON,J. E., J. Microsc. 19, 59 (1974). 14. FLI~CHON,J. E., in HAFEZ, E. S. E. (Ed.), Scanning Electron Microscope Atlas of Mammalian Reproduction, p. 68. Shoin, Tokyo, 1975. 15. FLECHON, J. E., in HAFEZ, E. S. E. AND THIBAULT, C. (Ed.), The biology of spermatozoa, transport, survival and fertilizing ability, Inserm Int. Symp. Nouzilly 36, Karger, Basel (1975).
16. FRANKLIN, L. E., in AFZELIUS, B. A. (Ed.), The Functional Anatomy of the Spermatozoon 89, Pergamon, Oxford, 1974. 17. FRANKLIN, L. E., BARROS, C., AND FUSSELL, E. N., Biol. Reprod. 3, 180 (1970). 18. FRANKLIN, L. E., AND FUSSELL, E. N., Biol. Reprod. 7, 194 (1972). 19. GORDON, M., Z. Zellforsch. Mikrosk. Anat. 131, 15 (1972). 20. JONES, R. C., in FAVARD, P. (Ed.), Congr. Int. Microsc. t~lectr., 7th, 3, 641 (1970). 21. JONES, R. C., J. Reprod. Fertil. Suppl. 13, 51 (1971). 22. LALLI, M. F., thesis, McGill University, Montreal, 1972; Dis. Abstr. 33, 2900 (1973). 23. MORSTIN, J., AND COUROT, M., Syrup. Int. Zootech., 8th (Milan) 870 (1973). 24. NICANDER,L., AND BANE, A., Z. Zellforsch. Mikrosk. Anat. 72, 496 (1966). 25. PLSEN, L., Z. Zellforsch. Mikrosk. Anat. 115, 553 (1971). 26. SANDOZ,D., J. Microsc. 9, 535 (1970). 27. SFURR, A. K., J. Ultrastruct. Res. 26, 31 (1969). 28. TEICHMAN, R. J., CUMMINS, J. M., AND TAKEL, G. H., Biol. Reprod. 10, 565 (1974). 29. THI~RY, J. P., AND RAMEOURG, A., J. Microsc. 21, 225 (1974). 30. TOKUYASU,K. T., J. Cell. Biol. 57, 551 (1973). 31. YANAGIMACHI,R., AND NODA, Y. D., J. Ultrastruct. Res. 31, 465 (1970).
(1976)
The Perinuclear Substance of Boar, Bull, Ram and Rabbit Spermatozoa J . L. COURTENS, 1 M. COUROT, 1 AND J. E. FLECHON ~
Station de Physiologie de la Reproduction, I.N.R.A., 37380 Nouzillyl; Station centrale de Physiologie anirnale, I.N.R.A., 78350 Jouy-en-Josas,2France Received February 10, 1976 The perinuclear substance (PNS) in boar, bull, ram and rabbit spermatozoa is described. The PNS is present as a thin, continuous layer of lysine-rich proteins located between the nucleus on one side and the acrosome and postacrosomal lamina on the other. The perforatorium and equatorial blisters (rabbit) are part of the PNS. PNS first appears when the acrosomal vesicle apposes on the nucleus of young spermatids. Morphological and cytochemical modifications of PNS occur during spermiogenesis and epididymal maturation. The functional role of this sperm cell entity is briefly discussed. L'~tude de la substance p~rinucldaire (SPN) est effectu~e dans les spermatozo~des de Verrat, Taureau, B~lier et Lapin. La SPN est constitute d'une fine couche de prot~ines riches en lysine, dispos~e de mani~re continue entre le noyau d'une part, l'acrosome et la lame postacrosomique d'autre part. Le perforatorium et les ampoules 6quatoriales (Lapin) font partie de la SPN. La SPN apparalt pour la premiere fois au moment de l'accolement de la v~sicule acrosomique sur le noyau des jeunes spermatides. Des modifications morphologiques et cytochimiques de la SPN se produisent au cours de la spermiogen~se et de la maturation ~pididymaire. Le rSle fonctionnel de cette entit~ du gamete m~le est discut~. ( P N S ) for al l t h e m a t e r i a l l o c a t e d b e t w e e n t h e n u c l e a r e n v e l o p e a n d t h e a c r o s o m e or p o s t a c r o s o m a l l a m i n a (3, 12, 23). Is t h e P N S a s t r u c t u r a l , b i o c h e m i c a l ent i t y ? T h e p u r p o s e of t h i s w o r k is to des c r i b e t h e d i s t r i b u t i o n of P N S , its o r i g i n a n d c y t o c h e m i c a l c o m p o s i t i o n i n t h e sp er m a t o z o a of c o m m o n f a r m a n i m a l s .
M o s t of t h e t r a n s m i s s i o n e l e c t r o n m i c r o scope s t u d i e s on t h e h e a d of m a m m a l i a n s p e r m a t o z o a h a v e d e m o n s t r a t e d the prese n c e of m a t e r i a l l o c a t e d b e t w e e n t h e acros o m e a n d t h e n u c l e a r e n v e l o p e , i n t h e soca]led s u b a c r o s o m a l space. S o m e a u t h o r s h a v e n o t r e c o g n i z e d t h e e x i s t e n c e of t h i s m a t e r i a l (10), or a t l e a s t i ts s i g n i f i c a n c e (11). O n l y J o n e s (20, 21) d e s c r i b e d a conn e c t i o n b e t w e e n t h e p e r f o r a t o r i u m (or apical body), t h e s u b a c r o s o m a l s u b s t a n c e a n d t h e m a t e r i a l p r e s e n t on t h e n u c l e a r e n v e lope u n d e r t h e p o s t a c r o s o m a l l a m i n a in ram and boar spermatozoa. Continuous p e r i n u c l e a r m a t e r i a l was also i n c i d e n t a l l y s h o w n i n s p e r m of g o l d e n h a m s t e r , r a b b i t , a n d t u p a i a (1, 17, 19). We s h a l l r e s t r i c t t h e t e r m s ~¢perforator i u m " a n d " s u b a c r o s o m a l s u b s t a n c e " to t h e i r c l a s s i c a l m e a n i n g s (24), a n d u s e t h e general term "perinuclear substance"
MATERIAL AND METHODS
Morphological S t u d i e s Ram testis and rabbit and ram epididymis were fixed by perfusion or immersion with glutaraldehyde fixative. Various combinations of glutaraldehyde in phosphate or cacodylate buffers were used for fixation of ejaculated (and uterine) sperm of boar, bull, rabbit, and ram. The material was embedded either in glycol methacrylate or, after eventual osmium tetroxide prefixation, in (agar and) Epon, in a mixture of Araldite and Epon or in Spurr's medium (27). Fixed sperm cells were also processed by ultracryotomy (30). Ultrathin sections were stained either with alcoholic or aqueous uranyl acetate and lead citrate or in alcoholic solution of phosphotungstic acid.
1J.L.C. and M.C. 2 J.E.F. 54 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
Cytochemical Studies of Protein Amino Residues Ram testis was fixed by immersion in 4% glutaraldehyde in 0.1 M phosphate buffer. After inhibiting sulfhydryl and guanidyl groups with n-ethyl maleimide and malonaldehyde, respectively (1 and 2%, pH 7.4, 4 hr), the tissue was stained en bloc by alcoholic phosphotungstic acid (PTA/e) or hydroquinone-ferricyanide-copper phosphotungstic acid (HQ-Fe) to locate lysine residues according to Courtens and Loir (7). In some cases no inhibition was done before PTA/e treatment. Ultrathin sections in the Epon-embedded material were observed without any post-staining. RESULTS
Morphology of the Perinuclear Substance in Mature Spermatozoa In all types of fixations and embeddings used and after nonspecific staining, the spermatozoa show a continuous layer of p e r i n u c l e a r substance (PNS) u n d e r the acrosome and the postacrosomal lamina. The aspect of this substance differs according to the head region. The acrosomal region. In front of the a n t e r i o r nuclear edge, a large a m o u n t of the PNS is in the shape of a wedge, previously referred to as the p e r f o r a t o r i u m or apical body (Figs. 1 and 2); it appears to follow the horseshoe shape of the m a r g i n a l t h i c k e n i n g (Fig. 3). Towards the posterior end of the a n t e r i o r segment, where the m a r g i n a l t h i c k e n i n g decreases, the PNS also fills the p e r i n u c l e a r space over the edge of the nucleus, b u t does not r e t a i n its wedge shape in t r a n s v e r s e sections (Figs. 4-7, 9). On the dorsal and v e n t r a l sides of the nucleus, the P N S appears as a thin l a y e r in the e n t i r e subacrosomal space (Figs. 1, 2, 4-7, 9, 10); it is t h i c k e r u n d e r the equato• rial segment, especially at its limit with the a n t e r i o r segment (Figs. 8, 10, 13). In rabbit spermatozoa, the P N S is very expanded in this location, i.e., it forms at least two blisters of spongious, or sometimes homogeneous, m a t e r i a l on both sides of the nucleus (Figs. 6 and 9). The postacrosomal region. The PNS in
55
the postacrosomal region is clearly contiguous with t h a t in the subacrosomal space, and a small p a r t of it also appears to surround the posterior rim of the equatorial segment (Figs. 13, 14, 16). The PNS does not completely fill the space between the nucleus and the postacrosomal l a m i n a and appears loosely connected to the l a t t e r (Figs. 11-16). On sagittal sections, the posterior part of the PNS appears s t r a i g h t and closely applied on a portion of thickened nuclear envelope; it is h e r e a f t e r referred to as the ~postnuclear band" (Figs. 12, 14, 15). The ends of the PNS and postacrosomal l a m i n a are pinched t o g e t h e r u n d e r the posterior ring w h e r e the p l a s m a m e m b r a n e m a k e s a groove in the direction of the nuclear envelope (Figs. 11, 14, 15). M a t u r e rabbit spermatozoa show clear spaces between the nucleus and the PNS in the postacrosomal region (Fig. 15). However, these spaces are not present in i m m a t u r e and u t e r i n e spermatozoa (Figs. 14 and 16).
Origin and Chemical Composition of P N S of Ram Spermatozoa After HQ-Fe staining, the PNS is visible between nucleus and acrosome (Fig. 17) as soon as the acrosomal vesicle adheres to the nucleus (stages 1-3) (5). The PNS extends and its electron density increases (Fig. 18) as the acrosome cap spreads over the r o u n d spermatid nucleus (stages 4-8). The postnuclear band appears in the posterior part of the subacrosomal space during stages 9 and 10 and r e m a i n s in this location from stages 9 to 12. It consists of two components which join to form a diss y m m e t r i c a l V pointing to the anterior of the head (Figs. 19 and 20). The densely stained P N S extends into the postacrosomal region d u r i n g stage 12 concomitantly with the m i g r a t i o n of the postnuclear band and the perinuclear ring. The postnuclear band slips down between the nuclear envelope and the plasma membrane, during which time both its arms
56
COURTENS, COUROT AND FLECHON
Q P
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
become apposed on the nearest of these membranes (Fig. 21). The postnuclear band later remains as a clearly defined belt around the posterior end of the nucleus, in front of the posterior ring; the external arm of the V vanishes, and the internal arm is then anteriorly covered by the postacrosomal lamina applied along the plasma membrane (Fig. 22). At the end of stage 12 the perinuclear ring disappears. From stage 13 to spermiation, the postnuclear band becomes very narrow and entirely covered by the postacrosomal dense lamina (Fig. 12). When arginine is blocked with malonaldehyde, only the postnuclear band is stained by en bloc alcoholic PTA in spermatids (Fig. 20) and testicular spermatozoa, whereas all the PNS reacts in spermatoz from cauda epididymis. All the results obtained are summarized in Table I. DISCUSSION
Morphological Entity of PNS The PNS, visualized by various techniques including ultracryotomy (this study) and freeze-fracturing (13), constitutes a continuous layer under the acrosome and postacrosomal lamina in all the paddle-shaped spermatozoa studied. No wide species differences appear in the morphology of different PNS regions. For example, the PNS is thicker at the limit of the anterior and equatorial seg-
57
ments in the spermatozoa of the four species. However, the external development of this part of the PNS into blisters [from the mature spermatid stage (25)] is unique to rabbit and hare (24). The fact that the PNS is an entity is also shown by its genesis. PNS is laid down in ram spermatids as soon as a subacrosomal space exists, and also in rat (22) and golden hamster (16, 18). During elongation and flattening of the spermatid nucleus, PNS accumulates all around the edge of the nucleus under the acrosome. It assumes a wedge shape (the perforatorium) only under the anterior part of the marginal thickening, after elongation of the corresponding subacrosomal space [before spermiation in rabbit (25)]. In the postacrosomal region, the PNS appears to be an extension of the already present subacrosomal substance, consecutive to the migration of the postnuclear band, first described as "anneau postnucl~aire" (7). To avoid confusion with the posterior ring, the term postnuclear band is to be added to the sperm nomenclature proposed by some of us (3). It does not seem important whether the postnuclear band is a part of the PNS or only its posterior boundary. Although appearing at the same time in ram [as in mouse (26)], the highly differentiated postacrosomal lamina (14) cannot be confused with the underlying PNS.
Fia. 1. Sagittal section in the anterior part of a bull sperm head from cauda epididymis. The PNS is continuously spread between the nucleus and the acrosome and forms a finger-like anterior projection, the perforatorium (P). Glut, E, U and Pb; × 100 000. Abbreviations for all figures: Sperm morphology: A: acrosome; AP: arrays of particles; AV: acrosomal vesicle; AS: anterior segment of the acrosome; ES: equatorial segment of the acrosome; CC: crystalline core (in marginal thickening); DC: dense core (in m a r g i n a l thickening); G: acrosomal granule; N: nucleus; NE: nuclear envelope; P: perforatorium; PAL: postacrosomal lamina; PNB: postnuclear band; PR: posterior ring. Techniques: PTA/e: phosphotungstic acid in ethanol; E: Epon; Glut: glutaraldehyde; GMA: glycol methacrylate; HQ-Fe: hydroquinone-ferricyanidecopper phosphotungstic acid; Pb: lead citrate; U: uranyl acetate. Fia. 2. Similar section of an ejaculated rabbit spermatozoon. The PNS, including the wedge-like perfora~ torium (P), is visible between the acrosome and the nuclear envelope (NE). Glut, agar E, U and Pb; × 100 000. Fro. 3. Anterior portion of a n ejaculated r a m spermatozoon sectioned approximately parallel to the flat surface of the nucleus (N). The perforatorium is situated between the nucleus and the tip of the acrosome containing paracrystalline m a t e r i a l ( ~" ~ ). Glut, E, U and Pb; × 60 000.
58
COURTENS, COUROT AND FLt~CHON
Nature and Uniformity of PNS Chemical Composition Starting from its deposition, the PNS is a proteinaceous material, as first suggested by Nicander and Bane (24); it is apparently devoid of sugar residues since we failed to stain it with the techniques of Thi~ry and Rambourg (29). As shown by HQ-Fe staining after use of inhibitors, lysine residues seem to gather in the acrosomal and forming postacrosomal regions of the PNS at the same time as lysine-rich histones leave the nucleus in a
wave, progressing towards its posterior end (7). The disappearance of nuclear pores, as described in mouse (26), is probably related to the chromatin condensation and the changes in the PNS. Alcoholic PTA stainability after malonaldehyde inhibition increases during epididymal maturation in the whole PNS (except in the already stained postnuclear band). This may indicate a reduction in the distance between protein lysine groups (7). In fact, a condensation in the PNS and a shortening of the perforatorium were described at the same time in boar spermato-
FIG. 4. Transverse section of an ejaculated bull sperm head in the caudal part of the anterior segment. The PNS is thin, except on the lateral edges of the nucleus where it occupies the free space under the marginal thickening. Glut, E, U and Pb; × 50 000. FIG. 5. Slightly oblique section of a rabbit sperm head in the anterior segment. The continuous PNS shows a finger-like tip, the perforatorium (P) on the more anterior side (larger marginal thickening) and a blunt extremity in a posterior location (opposite end of the section). Glut, agar E, U and Pb; × 45 000. FIG. 6. Transverse section of a rabbit sperm head in the posterior part of the anterior segment. The PNS is thick on the lateral edges and developed in two spongious blisters (B) on t h e ventral and dorsal sides. Glut, agar E, U and Pb; x 56 000. FIG. 7. Oblique section in the posterior extremity of the anterior segment and in the equatorial segment of a rabbit sperm head. The PNS is thin under the equatorial segment (ES), except on the lateral edges and especially at the contact with the anterior segment. Glut, agar E, U and Pb; × 45 000. FIG. 8. Sagittal section of'a boar sperm head at the limit of the two acrosomal segments (AS and ES). The PNS fills the subacrosomal space, larger in this location. Glut, ultrathin frozen section, U and Pb; x 96 000. FIG. 9. Transverse sections of a rabbit sperm head in the region of the blisters (B) whicl~/extend over the lateral ridge (B'). Between the blisters and on the other lateral ridge (top) the PNS is thick. Acrosome and PNS are stained, as the technique used may be specific for proteins. Glut, GMA, PTA/e on the thin section; × 60 000. FIG. 10. Sagittal sections of boar sperm heads. The PNS is continuous in the subacrosomal space and thicker at the limit of the two acrosomal segments (--*). Glut, agar E, PTA/e on the thin section; x 40 000. FIG. 11. Postacrosomal region of a boar sperm head. The different stained layers from outside to inside are: plasma membrane, postacrosoma] lamina, PNS, nuclear envelope. As in the preceding picture, the staining used on sections has no known specificity, but outlines the PNS very well. Glut, .agar E, U in methanol and Pb; x 50 000. Fro. 12. Sagittal section of a ram testicular sperm head. The PNS appears to end posteriorly in a dense layer or postnuclear band (PNB) located between the postacrosomal lamina (PAL) and the nuclear envelope (NE). Glut, n-ethyl maleimide + malonaldehyde, HQ-Fe, E; x 100 000. FIG. 13. Sagitta] section of a bull sperm head from cauda epididymis: the PNS is contiguous under the acrosome and postacrosomal lamina (PAL); it is thicker at the limit of the equatorial segments (AS and ES) and surrounds the posterior end of the ES. Glut, E, U and Pb; × 60 000. FIG. 14. Sagittal section of a rabbit sperm head from corpus epididymis: contiguity of the PNS under the equatorial segment (ES) and the postacrosomal lamina (PAL). The PNS is caudally ended by the postnuclear band (PNB) inserted between the nuclear envelope (NE) and a thickened portion of the postacrosomal lamina, in front of the posterior ring (PR). Glut, E, U and Pb; × 65 000. FIG. 15. Similar section of an ejaculated rabbit sperm; a clear space (~-) is visible between the nucleus and the PNS under the postacrosomal lamina (PAL), except in the anterior and posterior extremities, The postnuclear band (PNB) is still applied on the nuclear envelope. Glut, agar E, U and Pb; x 84 000. FIG. 16. Rabbit spermatozoon recovered in the uterus 12 hr after coitus. The morphology is the same as for immature sperm. The postnuclear band is not visible in this slightly oblique section. Glut, E, U and Pb; × 64 000.
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
59
• PNS
PNS
Q
Q
AS
PNS
(9
60
COURTENS, COUROT AND FL~CHON
B'
PERINUCLEAR SUBSTANCE OF SPERMATOZOA
~®
61
®
62
COURTENS, COUROT AND FL~CHON
63
PERINUCLEAR SUBSTANCE OF SPERMATOZOA TABLE I CYTOCHEMISTRY OF PNS = PTA/e
PTA/e (after malonaldehyde blockade)
= HQ-Fe
Testis
+
-
+
Cauda epididymis
+
+
+
Groups of 7-8. Lys in prox.
Lysine
Ejaculated spermatozoa
+ (on thin sections in GMA)
Nature of constituents
Arginine and lysine
zoa (21). Finally, disulfide bonds, already present in the spermatid PNS (6), also increase during epididymal maturation (4). Such crosslinked proteins are relatively resistant to pronase extraction on thin sections of rabbit spermatozoa (15).
Role of the PNS The fact that the PNS appears as a structural and biochemical entity also suggests t h a t it plays a functional role. This role may be important for fertilization, as the PNS is the underlying substrate of the acrosome and postacrosomal lamina, the sperm elements which are related to egg penetration and attachment. As pointed out previously (9, 12, 16, 23), the PNS may act as a cement between the nucleus and the overlaying elements of the sperm head. In the postacrosomal region, the postacrosomal lamina and PNS are spatially linked, since in m a t ur e rabbit spermatozoa their mutual opposition is
not d i s t u r b e d by the presence of clear spaces over the nucleus. These spaces, reported to contain phospholipid material (28), disappear in uterine spermatozoa and the PNS returns close to the nuclear surface. In the acrosomal region the cement hypothesis is substantiated by the persistence of the inner acrosomal membrane on spermatozoa deprived of the anterior segment (2) and in the process of ovum penetration (7, 17, 31). The scleroprotein nat ure of the PNS may also give rigidity to the perforatorium, thus facilitating penetration of the zona pellucida (31). The technical assistance of D. Huneau is gratefully acknowledged. REFERENCES 1. BEDFORD,J. M., Contribution Primatology 3, 97 (1974). 2. BROWN, C. R., ANDANI, Z., AND HARTREE, E. F.,
FIG. 17-22. Spermiogenesis in the ram (glut, n-ethyl maleimide + malonaldehyde, HQ-Fe, E, except Fig. 21: PTA/e instead of HQ-Fe). FIG. 17. At stage 3, the stained PNS is visible between the dense peripheral chromatin and the membrane ( $ ) of the acrosomal vesicle (AV). × 53 000. FIG. 18. At stage 6, a thicker layer of PNS is interposed between the nucleus and the acrosomal vesicle containing an acrosomal granule (G). × 38 000. Fro. 19. Elongated spermatid (stage 11); the PNS is about as thick and stained as the acrosome (A). In front of the perinuclear ring (--~), both show a thickened end, V-shaped in the case of the PNS: the future postnuclear band (PNB). x 43 000. Fia. 20. The postnuclear band (PNB) appears as V-shaped stained material located in front of the perinuclear ring (-~), and between the posterior extremity of the acrosome (A) and a thickened portion of nuclear envelope (NE). In front of the postnuclear band, the PNS is not stained (stage 10). x 43 000. FIG. 21. During the sliding of the perinuclear ring (--~), the postnuclear band (PNB) follows the former. x 34 000. Fro. 22. At the end of stage 12, the perinuclear ring (-->) and postnuclear band (PNB) have reached the posterior part of the nucleus. PNS has deposited under the postacrosomal lamina (PAL). × 35 000.
64
COURTENS, COUROT AND FL~CHON
Biochem. J. 149, 133 (1975). 3. BUSTOS-OBREGON,E., COUROT, M., FL~CHON, J. E., HOCHEREAUDE REVIERS, M. J., AND HOLSTEIN, A. F., Andrologia 7, 141 (1975). 4. CALVIN, H. I., AND BEDFORD, J. M., J. Reprod. Fertil. Suppl. 13, 65 (1971). 5. CLERMONT, Y., AND LEBLOND, C. P., Arner. J. Anat. 96, 229 (1955). 6. COURTENS,J. L., unpublished data (1975). 7. COURTENS,J. L., AND LOIR, M., J. Microsc. Biol. Cell. 24, 249 (1975). 8. COURT~NS,J. L., AND LOIR, M., J. Microsc. Biol. Cell. 24, 259 (1975). 9. FAWCETT, D. W., Biol, Reprod. Suppl. 2, 90 (1970). 10. FAWC~TT, D. W., AND ITO, S., Amer. J. Anat. 116, 567 (1965). 11. FAWCETT, D. W., AND PHILLIPS, D. M., J. Reprod. Fertil. Suppl. 6, 405 (1969). 12. FLI~CHON, J. E., thesis, University of Paris, 1973. 13. FLI~CHON,J. E., J. Microsc. 19, 59 (1974). 14. FLI~CHON,J. E., in HAFEZ, E. S. E. (Ed.), Scanning Electron Microscope Atlas of Mammalian Reproduction, p. 68. Shoin, Tokyo, 1975. 15. FLECHON, J. E., in HAFEZ, E. S. E. AND THIBAULT, C. (Ed.), The biology of spermatozoa, transport, survival and fertilizing ability, Inserm Int. Symp. Nouzilly 36, Karger, Basel (1975).
16. FRANKLIN, L. E., in AFZELIUS, B. A. (Ed.), The Functional Anatomy of the Spermatozoon 89, Pergamon, Oxford, 1974. 17. FRANKLIN, L. E., BARROS, C., AND FUSSELL, E. N., Biol. Reprod. 3, 180 (1970). 18. FRANKLIN, L. E., AND FUSSELL, E. N., Biol. Reprod. 7, 194 (1972). 19. GORDON, M., Z. Zellforsch. Mikrosk. Anat. 131, 15 (1972). 20. JONES, R. C., in FAVARD, P. (Ed.), Congr. Int. Microsc. t~lectr., 7th, 3, 641 (1970). 21. JONES, R. C., J. Reprod. Fertil. Suppl. 13, 51 (1971). 22. LALLI, M. F., thesis, McGill University, Montreal, 1972; Dis. Abstr. 33, 2900 (1973). 23. MORSTIN, J., AND COUROT, M., Syrup. Int. Zootech., 8th (Milan) 870 (1973). 24. NICANDER,L., AND BANE, A., Z. Zellforsch. Mikrosk. Anat. 72, 496 (1966). 25. PLSEN, L., Z. Zellforsch. Mikrosk. Anat. 115, 553 (1971). 26. SANDOZ,D., J. Microsc. 9, 535 (1970). 27. SFURR, A. K., J. Ultrastruct. Res. 26, 31 (1969). 28. TEICHMAN, R. J., CUMMINS, J. M., AND TAKEL, G. H., Biol. Reprod. 10, 565 (1974). 29. THI~RY, J. P., AND RAMEOURG, A., J. Microsc. 21, 225 (1974). 30. TOKUYASU,K. T., J. Cell. Biol. 57, 551 (1973). 31. YANAGIMACHI,R., AND NODA, Y. D., J. Ultrastruct. Res. 31, 465 (1970).
