THE JOURNAL OF EXPERIMENTAL ZOOLOGY 256~315-322 (1990)

Studies on the Decondensation of Human, Mouse, and Bull Sperm Nuclei by Heparin and Other Polyanions S. JAGER, J. WIJCHMAN, AND J. KREMER Fertility Unit, Department of Obstetrics and Gynaecology, University Hospital, 9713 EZ Groningen, The Netherlands ABSTRACT We report heparin-induced decondensation of human, mouse, and bull sperm nuclei. Decondensation did not occur if the spermatozoa were intact but only if the membranes were severely damaged by freezing and thawing or by treatment with a detergent. If a disulphide bond reducing agent (thiol) was absent, decondensation of human sperm nuclei was usually a relatively slow process, with large interindividual variation. Mouse and bull sperm nuclei did not decondense in the absence of a thiol. With a thiol relatively low concentrations of heparin induced a rapid decondensation of the sperm nuclei of all three species. The decondensation activity was not specific for heparin; other polyanions were also active, with heparin being the most effective compound. It is supposed that heparin and other polyanions induce sperm nuclear decondensation because they deplete protamines from the chromatin. Thus the negatively charged phosphate groups of the DNA are no longer opposed by positively charged protamines. Consequently the mutual repulsion of unopposed phosphate groups causes the DNA molecules to stretch, which results in a n increase of the sperm nuclear volume. Since heparin and other polyanions induce decondensation under physiological pH and temperature, polyanions might also be active in the oocyte.

The nucleus of the mammalian spermatozoon becomes highly condensed during spermatogenesis, which is due t o exchange of the histones by the more basic arginine-rich protamines. In eutherian mammals the adjacent protamine molecules become extensively cross-linked by disulphide bonds, mainly during passage through the epididymis (Calvin and Bedford, '71; Calvin et al., '73). This cross-linking endows the mature sperm nucleus with an extraordinary mechanical and chemical resistance (Bril-Petersen and Westenbrink, '63). Despite this high resistance the spermatozoa1 nucleus decondenses rapidly after incorporation into the oocyte (Austin, '61; Barros and Franklin, '68; Yanagimachi and Noda, '70). It is likely that this decondensation process in vivo involves disulphide bond reduction, which, according to Wiesel and Schultz ('€411,Calvin et al. ('861,Perreault et al. ('88b), may be due to reduced glutathione in the ovum. In vitro studies, however, show that an additional factor is required. The additional factors used in experiments were either a detergent (Calvin and Bedford, '71; Mahi and Yanagimachi, '75), a protease (Marushige and Marushige, '75; Gall and Ohsumi, '76; Young, '79)' or a high salt concentration (Gall and Ohsumi, '76; Rodman et al., '82). The nature of the additional decondensation factor, present in vivo, is not yet known. Ejaculated spermatozoa of man, in contrast t o those of other mammals, do not all require disul0 1990 WILEY-LISS, INC.

phide bond reduction t o decondense in vitro. Without a thiol a proportion of human spermatozoa was found to decondense in the presence of the detergent sodium dodecyl sulphate (SDS) at high pH (Bedford et al., '73). Recently it has been reported that in the absence of a thiol heparin can induce nuclear decondensation of human ejaculated spermatozoa, but not of ejaculated spermatozoa from bull, pig, rat, or rabbit (Delgado et al., '80; Caranco et al., '83). Since heparin is a naturally occurring substance and since the in vitro decondensation occurred under physiological temperature and pH, heparin might act in vivo as well (Reyes et al., '89). In the present study the heparin-induced decondensation of sperm nuclei is investigated. The possible mechanisms of the in vitro decondensation and its occurrence in the ovum are discussed.

MATERIALS AND METHODS Spermatozoa Human ejaculated spermatozoa were obtained from normal donors, from normospermic men visiting the fertility unit, and also from a man with round-headed spermatozoa. Ejaculates were collected by masturbation into a wide-mouth plastic jar and delivered at the laboratory within 2 hours after ejaculation. The spermatozoa were Received May 10, 1989; revision accepted April 6, 1990.

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isolated either by washing with 0.1 M glycine/ NaOH buffer or by migration. In the washing procedure the seminal fluid was centrifuged for 10 min at 600g. The sediment was suspended in 0.2 ml 0.1 M glycine/NaOH buffer, pH 7.2, unless otherwise stated. In the migration procedure 0.2 ml semen was pipetted into a small, round-bottom plastic tube (9 mm diameter). Upon the seminal fluid 0.3 ml Earle's balanced salt solution (EBBS) containing 10 g/L bovine serum albumin (BSA) was carefully layered. After 1-2-hour incubation at 37°C 0.2 ml EBBS containing 40 g/L albumin was carefully added, Thus, a clear interphase separated the seminal fluid from the upper layer containing migrated spermatozoa of which over 90% showed a good forward motility. This interphase allowed collection of all the migrated spermatozoa with minimal contamination by seminal plasma. The migrated spermatozoa were used either untreated or after storage at - 70°C. Bull-ejaculated spermatozoa were collected by standard techniques and stored in French straws at - 196°C. They were kindly supplied by the KIVereniging GroningedNoord Drente, The Netherlands. After thawing, the spermatozoa were blown out into 0.1 M glycinelNaOH buffer, and centrifuged 5 times with this buffer. The sediments were stored at -20°C. Mouse spermatozoa were obtained from mice from a heterogeneous strain, produced by crossing C3H, C57/BL, and DBA. The animals were killed with C02 gas, and the vas deferens was removed. The spermatozoa were squeezed from the vas into the glycine/NaOH buffer. The suspension was washed 3 times with this buffer and the sediment was stored at - 20°C. The spermatozoa from all three species were, after at least 1 day, thawed and frozen again in order to disrupt the membranes, and stored at - 20°C. After rethawing, all spermatozoa stained red with eosin Y in a supravital staining method (Eliasson and Treichl, '71; Jager et al., '841, which indicated that the membranes from all spermatozoa were disrupted.

Decondensation experiments Decondensation experiments were performed by adding 0.1 ml of heparin (approximately 170 USP units/mg) (Organon-Diosynth, Oss, The Netherlands) in 0.1 M glycine/NaOH buffer, pH 7.2, t o a sediment of spermatozoa to a final concentration of lo5 spermatozoa per ml. The buffer contained up to 0.5 M dithiothreitol (DTT) and/or 1%(v/v) Triton X-100. To investigate the specifi-

city of heparin- induced decondensation, various other heparin preparations (Sigma) were also tested: ammonium salt (140 USP unitdmg), calcium salt (140 'IJSP units/mg), lithium salt (160 USP unitdmg), sodium salt (grade 11, 140 USP units/mg), sodium salt (low molecular weight, average 4,000-6,300), sodium salt (crude unbleached, not less than 70 USP unitdmg), all from porcine mucosa. In other experiments the heparin was replaced by other polyanions: chondroitine sulphate types A, B, C (Sigma); dextran sulphate, molecular weight approximately 5,000 (Sigma); dextraii sulphate, molecular weight approximately 500,000 (Serva); polyadenylic acid (Sigma); ribonucleic acid (RNA) from baker's yeast (Merck);polygalacturonic acid (Sigma); and hyaluronic acid (Sigma).

Assa,ys of decondensation The decondensation of sperm nuclei was usually determined by counting the spermatozoa with a swollen appearance in a wet preparation under an interference contrast microscope at 12.5 x 25 magnification. In other experiments a photographic method was used. Every 15-30 sec slides were made of the same optical field. The slides were prqlected onto a piece of paper, the outlines of the sperm heads marked with a pencil, and their surfaces measured with a planimeter. The nuclear swelling was also visualized by staining with Giemsa (Fig. 2). A droplet of a suspension with swelling spermatozoa was applied onto a slide. The slide was dipped immediately into methanol and air-dried after 15 min. The preparation was stained by immersing the slide for 30 min in an 0.067 M phosphate-buffer, pH 6.9, with a Romanovski-Giemsa solution (16%, v/v). The slide was rinsed thrice in tapwater.

Assag and inhibition of proteolytic activitg Proteolytic activity in the spermatozoa was detected with a gelatin-nigrosin method. Two g gelatin, 1 g nigrosin, and 0.42 g sodium-cacodylate were dissolved in 20 ml boiling water. When the suspension was cooled down to 60"C, aliquots of 0.2 In1 were pipetted into small plastic tubes at room temperature that contained 0.05 ml of a spermatozoa suspension in EBBS with 1% (v/v>Triton X-LOO and with or without 0.05 g/L soybean trypsirl inhibitor. Immediately after mixing, a drop was pipetted onto a microscopic slide. A coverslip was laid on the mixture and pressed slightly to obtain a thin layer. After the preparations were stored at room temperature for 6 hours

HEPARIN-INDUCED SWELLING

in a moist Petri dish the slides were observed under a light microscope. Proteolytic activity of the spermatozoon causes digestion of the gelatin, which results in a clear area (halo) around the sperm head. To exclude involvement of protease activity commercially available protease inhibitors were used: Aprotinin (0.14 mM), Bestatin (0.3 mM), E64 (0.06 mM), Leupeptin (0.2 mM), pepstatin (0.004 mM), Phenantrolin (1 mM), PMSF (2.0 mM), TLCK (1.43 mM). In addition EDTA was used (20 mM). All compounds were used without further purification.

RESULTS Decondensation in the absence of a thiol The heads of washed human spermatozoa swelled in a medium containing 60 g/L heparin (Organon) and no DTT, with a swelling rate often increasing in time. However, a suspension of washed human ejaculated spermatozoa is always a mixture of live and dead cells. To investigate whether either the living or the dead spermatozoa swelled or both, we isolated motile spermatozoa with the migration method. Over 90% of these migrated spermatozoa showed good forward motility and were thus intact. Up to 5 hours almost none of these spermatozoa swelled in the presence of heparin (Fig. 1).The supravital staining percentage increased from 15% to 67%, which demonstrates that at the end of the incubation time the majority of the spermatozoa were dead and had damaged cell membranes. Another portion of the migrated spermatozoa was first frozen-thawed twice. The frozen-thawed spermatozoa started swelling immediately upon addition of the heparin, and after approximately 1 hour some 50% of the sperm heads showed signs of swelling. These results show that heparin cannot induce nuclear decondensation of intact spermatozoa. Frozen-thawed spermatozoa swelled approximately twice as rapidly at 37°C as at room temperature. From one donor 50% of the spermatozoa swelled in about 40 min at room temperature (22°C) and in about 20 min at 37°C. With spermatozoa from another donor these 50% swelling times were 35 min and 15 min. Swelling rates of frozen-thawed spermatozoa showed great variations. Within one ejaculate various swelling stages could be seen at the same time (Fig. 2). Between individuals a strong variation was also observed: 10% to 100% swollen sperm heads after 1 hour. Frozen-thawed sper-

2

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Fig. 1. Time course of decondensation of human sperm nuclei in the presence of 60 mg/L heparin. No thiol was added. Spermatozoa from a donor semen sample were isolated by centrifugation and resuspension ( 0 )or by migration. The migrated spermatozoa were used either without further treatment (A)or after freezing and thawing (0). The percentages of swollen sperm nuclei were determined in wet preparations by counting.

matozoa from one man even showed 100% swelling within a few minutes after the heparin was added. This observation was confirmed with two other semen samples from the same man. We also tested round-headed spermatozoa known t o be devoid of an acrosome and therefore t o lack the acrosomal protease. Since the motility was not sufficient for migration, we tested washed spermatozoa. About 50% of the sperm nuclei swelled within 30 min. Thus the heparin-induced swelling does not involve acrosomal proteolytic activity.

Decondensation in the presence of a thiol Frozen-thawed human sperm heads swelled much more rapidly if 0.05 M DTT was present in addition to the heparin (Fig. 3). The swelling started immediately, and within a few minutes the sperm heads became invisible. A rapid swelling was also observed with much lower concentrations. The concentration needed to induce swelling of all sperm nuclei in about 5 min, using spermatozoa from 5 donors, varied from 0.3 g/L to 0.03 g/L. Lower concentrations could not be used

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50 70

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Fig. 2. Heterogeneity of swelling of human sperm nuclei induced by heparin in the absence of a disulphide bond reducing agent. Preparation stained with Romanovski-Giemsa. x 625.

since the sperm nuclei no longer swelled but disintegrated. The thiol-dependent swelling was so Fig. 3. Time coLrse of decondensation of human sperm rapid that swelling rates could not be determined nuclei in the presence of 60 mgiL heparin and 0.05 M dithioby counting. Therefore, for establishing the dose- threitol (DTT). The i;ame optical field was photographed variresponse relation we estimated the time at which ous times. The imag?s of the sperm nuclei were projected on a piece of white paper and the surfaces of sperm nucleus proall sperm nuclei showed signs of swelling with jections measured. ?'he means and standard deviations of the various heparin concentrations. However, in view same six nuclei are presented. Zero decondensation was deof the strong interindividual variation, as indi- termined from a spermatozoa suspension without heparin cated before, a precise dose-response curve could and without DTT. not be established. An example of a dose-response relation thus obtained is presented in Figure 4. 9/1 Untreated human migrated spermatozoa did not swell with heparin in the presence of a thiol; O ,' r 7 but if the detergent Triton X-100 was also present a rapid swelling occurred. Without heparin a slow swelling of the sperm heads was seen in the pres10ence of Triton X-100; after 4 hours 10% t o 50% of the sperm heads were swollen. The swelling was observed with spermatozoa suspended in an 0.05 M Tris/HCl, pH 7.3, buffer but not if the glycine 1buffer was used. This slow thiol-dependent, heparin-independent swelling, which was inhibited considerably with 2.0 mM PMSF, can probably be ascribed t o acrosomal proteolytic activity. Accordingly, we found haloes around the heads of all 0;r migrated spermatozoa in the gelatin-nigrosin preparations, but not if trypsin inhibitor was present. No haloes were seen around heads of frozen-thawed spermatozoa, which indicates that the 5 10 freezing and thawing removed the acrosomal prominutes tease. Fig. 4. Dose-response curves of decondensation of human None of the protease inhibitors decreased the spermatozoa in the presence of various polyanions and 0.05 M rate of the heparin-dependent swelling even with dithiothreitol (DTT). Y-axis: concentration in giL, logaritha heparin concentration as low as 1g/L. Thus, it is mic scale; X-axis: time a t which almost all sperm heads were unlikely that the heparin-induced swelling in- swollen. a: heparin, b: dextran sulphate MW 5,000; c: chondroitin sulphate type B; d: dextran sulphate MW 500,000. volves proteolytic activity. I

1

1

1

1

I

1

I

I

I

HEPARIN-INDUCED SWELLING

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TABLE 1. Minimal concentrations of heparin and other polyanions required to induce within 5 minutes approximately 100% swellirtg of sperm nuclei in a solution of 0.05 M (human and mouse) or 0.5 M ( b i d ) ~ i t h i o t h r e i t at ~ l pH 7 8 Concentration (giL) Mouse Human

Polyanion

Bull

Heparin Chondroitin sulphate type A Chondroitin sulphate type B Chondroitin sulphate type C Dextran sulphate MW 5,000 Dextran sulphate MW 500,000 Polyadenylic acid RNA Hyaluronic acid Galacturonic acid

301 25 >301 3 30 30 25 >15l >301

3 >301 15 >301 3 >301 30 25 >15l >301

301 301 3 30 30 8 >15l >301

'Maximal concentration tested and no or little swelling.

If DTT was present in addition to the heparin, the swelling rate of frozen-thawed sperm heads of bull and mouse was approximately as high as that of human sperm heads. However, a ten times higher concentration of DTT (0.5 M) was required for bull spermatozoa.

Effects of various polyanions To investigate the specificity of the heparininduced swelling we tested several heparin preparations and various other polyanions. Without a thiol all heparin types induced swelling of human sperm nuclei with the Organon heparin being the most effective and the ammonium salt heparin the least effective (swelling rate four times slower than the Organon preparation). The magnitude of the swelling rate between individuals varied greatly as indicated before. But the order of effectivity of the various heparin types was approximately the same with sperm nuclei of all six donors. The preparations with the highest specific activity induced the swelling most effectively. In the presence of 0.05 M DTT a rapid swelling of human sperm nuclei was seen not only with heparin but also with other polyanions (Table 1). The swelling of human sperm nuclei using dextran sulphate LMW was approximately as effective as heparin (Organon), whereas dextran sulphate HMW was two or three times less effective (Fig. 4). In the presence of a thiol the spermatozoa of bull and mouse showed a similar reaction to heparin but swelled uniformly t o a small extent only with dextransulphate, RNA, and chondroitin-B (Table 1). A solution of 30 t o 60 g/L of each of the tested substances (except hyaluronic acid, 15 g/L)

mixed with an equal volume of 6 glL salmon protamine instantaneously gave a white bulky precipitate. All polyanions thus had a strong affinity t o the protamine.

DISCUSSION We found that the proportion of human sperm nuclei decondensing with heparin, independent of a thiol, showed a strong intra- and interindividual variation. Similar observations were made with SDS by Bedford et al. ('731, who also found a corresponding heterogeneity of chromatin appearance in ultrastructural studies. These authors explained their observations by the very short and highly variable transit time of spermatozoa through the human epididymis (Rowley et al., '70; Amann and Howards, '80). In the epididymis the condensation of the spermatozoa1 chromatin is completed by establishing -S-Scross-links (Calvin and Bedford, '7 1). Consequently in human spermatozoa the number of intranuclear disulphide bonds may be low and highly variable. The interspecies differences in sperm nuclear stability, reported previously (Bedford et al., '73; Mahi and Yanagimachi, '75; Perreault et al., '88a) and confirmed once more in the present paper, have also been related to number and/or arrangement of disulphide bonding (Perreault et al., '88a). This in turn is determined by the type of protamine(s) present in the sperm nucleus. Bull sperm nuclei contain only protamine I (Calvin, '761, which is maximally crosslinked by disulphide bridges (Balhorn, '821, and are, therefore, probably very stable. Human and mouse sperm nuclei, on the other hand, contain protamine I1 as well as protamine I (Calvin, '76;

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Balhorn et al., '84;McKay et al., 1985, 1986). Since protamine I1 contains less cysteine, mouse and human sperm nuclei can be expected to have a lower concentration of disulphide bridges and thus t o be less stable than bull sperm nuclei. The variable stability of human sperm nuclei might also be related to variations in ratio of protamine I and protamine 11. Another explanation has been offered by Kvist et al. ('801, who suggested that the zinc content of sperm nuclei may play a role in sperm nuclear stability. However, their results were obtained with washed ejaculated human spermatozoa, these being a mixture of live and dead cells. As a consequence their results might also be explained by a zinc accumulation into or zinc depletion of dead spermatozoa, while it remains to be demonstrated that zinc can pass the plasma membrane of intact spermatozoa. Bedford et al. ('73), using SDS, already observed a decreased sperm nuclear stability in infertile men. Eliasson and Enquist ('81) proposed the use of SDS to test the stability of human sperm nuclei. Since we consider it likely that a heparinlike substance may be a decondensing factor present in the oocyte, we believe that the use of heparin for that aim is to be preferred. Our results show that heparin cannot induce thiol-independent swelling of intact human spermatozoa, as supposed by Caranco et al. ('83). Intact spermatozoa do not swell, even if incubated for hours. Even when the cell membranes are damaged such that supravital staining molecules can leak into the cells, heparin does not necessarily induce swelling of the sperm nucleus. Only if the membranes (and cytoplasm?) are severely damaged, either by freezing and thawing or by the action of detergents, will heparin induce a sperm head swelling immediately. This suggests that heparin acts directly on the chromatin mass, which may also be the case for the decondensation factors in the oocyte. In the oocyte, sperm nuclei decondense only after completion of the acrosome reaction and fusion of the oolemna with the sperm membrane overlying the equatorial segment (Yanagimachi and Noda, '70; Sathanantan et al., '86). Thus, the ooplasma is in close contact with the sperm nucleus without membranes in between. We therefore consider it possible that a heparinlike compound may play a role in the sperm nuclear decondensation within the oocyte, a concept supported by the observation that follicular fluid contains considerable amounts of glycosaminoglycans (Bellin et al., '86). In addition, rat ovary has been found t o synthesize heparinlike gly-

cosaminoglycans in vitro (Gebauer et al., '78). Furthermore, decondensation of human and sea urchin sperm nuclei could be induced with glycosaminoglycans extracted from sea urchin eggs (Delgado et al., Finally, it has now been demonstrated that niouse and bull spermatozoa can also decondense with heparin. The heparin-induced swelling is probably due to a direct reaction of heparin with the chromatin, since the swelling starts immediately upon addition of the heparin, provided the sperm nucleus is exposed, either by freezing and thawing or by treatment with a detergent. Should heparin induce the swelling indirectly, for instance, by activating a protease, then a lag phase would be expected. In addition, protease inhibitors did not decrease the swelling rate. The swelling process is not specific for heparin, since other polyanions also cause sperm nuclear decondensation. Previously, Dean ('83) demonstrated that polyglutamic acid could cause a decondensation of mouse sperm nuclei pretreated with dithiothreitol and with iodoacetamide to alkylate the reducled SH groups. Presumably, heparin, which is a polyanion, competes with DNA for binding to protamines. Heparin is known to have a strong affinity to protamine molecules; Chargaff and Olson ('38) ,already demonstrated that heparin and protamine molecules combine to an insoluble complex. Since the strong condensation of the sperm nucleus is the result of binding of protamines to the DNA, dissociation of the protamine-DNA complex will lead to decondensation of the chromatin and thus to an increase in the nuclear volume. The cause of t'ne dissociation of the protaminesDNA complex cmld be a coprecipitation of polyanions and protamines. According to this assumption, a polyaniori can cause decondensation only if the affinity of that polyanion to the protamines is higher than that of the DNA to the protamines. The differences in effectiveness between the various types of heparin and the various polyanions (Table 1)may thus reflect differences in affinity t o protamines. These differences may be ascribed to the charge density and the strength of the anionic groups. Sulphated polyanions, which have strong acidic groups, can be expected to be more effective than the non-sulphated polyanions hyaluronic acid and polygaJacturonic acid. The negative results with the types A and C chondroitin sulphate preparations might be explained by differences in either conformation or charge density or both. Of the many glycosaminoglycans found in tissues, '€12).

HEPARIN-INDUCED SWELLING

heparin has one of the highest sulphate contents per disaccharide unit. Similarly, swelling can be induced by SDS, a charged detergent, but not by neutral detergents (Mahi and Yanagimachi, '75; Jager et al., '83): only SDS precipitates protamines (Colom and Subirana, '79). Previously we supposed that SDS caused nuclear decondensation as a consequence of denaturation of nuclear proteins (Jager et al., '83). Dissociation of the protamines-DNA complex must also be the cause of nuclear decondensation in the presence of a high salt concentration, since Rodman et al. ('82) demonstrated with mouse sperm nuclei that under these conditions the protamines were extracted from the nuclei. Another decondensation mechanism has been suggested by Marushige and Marushige ('751, who presented evidence that the acrosomal enzyme acrosin was involved. Acrosin is probably also the cause of the thiol-dependent, heparinindependent swelling of migrated human spermatozoa treated with Triton X-100. This mechanism has been demonstrated by Young ('79) with rabbit spermatozoa. However, Uhehara and Yanagimachi ('76) demonstrated that freezethawed hamster and human spermatozoa retain their ability to decondense after injection into the hamster egg. In the present paper we showed that freeze-thawed spermatozoa are devoid of proteolytic activity and that round-headed spermatozoa, lacking acrosin (Wolff et al., '76; Lalonde et al., '82), do swell in the presence of heparin. Besides, Syms et al. ('84)found that round-headed spermatozoa can swell in crushed zona-free hamster ova, and Perreault and Zirkin ('82) showed that protease-free sperm heads decondensed after microinjection into eggs. We therefore consider it unlikely that an acrosomal proteolytic enzyme is responsible for the decondensation of the sperm nucleus in vivo. But the involvement of a proteolytic enzyme in the ooplasm with a very restricted specificity cannot be excluded yet. It has also been suggested that sperm nuclear decondensation in the oocyte may be induced by a local increase in ionic concentration, since in vitro decondensation can be induced by a high salt concentration (Rodman et al., '82). We consider such a mechanism unlikely, since it is difficult to envisage how this could occur in the ooplasm of crushed zona-free hamster ova (Syms et al., '84). Another possible decondensation mechanism in the oocyte might be phosphorylation of the protamines, as suggested by Young ('79). Phosphorylated protamines appear to have a decreased af-

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finity t o DNA (Willmitzer et al., '77a'b). Indeed a high level of protein phosphorylation has been demonstrated in mouse eggs soon after fertilization (Young and Sweeny, '78). Furthermore, the existence of a protein kinase has been demonstrated in unfertilized rabbit eggs (Wiesel and Schulz, '81).However, it remains to be demonstrated that the protamines are already being phosphorylated while still present in the chromatin. The phosphorylation might well be preceded by a polyanion induced swelling of the sperm nucleus.

LITERATURE CITED Amann, R.P., and S.S. Howards (1980) Daily spermatozoal production and epididydmal spermatozoal reserves of the human male. J. Urol., 124:211-215. Austin, C. (1961) The Mammalian Egg. Thomas, Springfield, IL. Balhorn, R. (1982) A model for the structure of chromatin in mammalian sperm. J. Cell Biol., 93:298-305. Balhorn, R., S. Weston, C. Thomas, and A. Wyrobek (1984) DNA packaging in mouse spermatids; synthesis of protamine variants and four transition proteins. Exp. Cell Res., 150:298-308. Barros, C., and L.E. Franklin (1969) Behavior of the gamete membranes during sperm entry into the mammalian egg. J Cell Biol., 37:C13-C18. Bedford, J.M., M.J. Bent, and H.I. Calvin (1973) Variations in the structural character and stability of the nuclear chromatin in morphologically normal human spermatozoa. J. Reprod. Fertil., 33:19-29. Bellin, M.E., R.L. Ax, N. Laufer, B.C. Tarlatzis, A.H. DeCherney, D. Feldberg, and F.P. Haseltine (1986) Glycosaminoglycans in follicular fluid from women undergoing in vitro fertilization and their relationship to cumulus expansion, fertilization, and development. Fertil. Steril., 45: 244-248. Bril-Petersen, E., and H.G.K. Westenbrink (1963) A structural basic protein as a counterpart of deoxyribonucleic acid in mammalian spermatozoa. Biochim. Biophys. Acta, 76: 152-154. Calvin, H.I. (1976) Comparative analysis of the nuclear basic proteins in rat, human, guinea pig, mouse and rabbit spermatozoa. Biochim. Biophys. Acta, 434:377-389. Calvin, H.I., and J.M. Bedford (1971) Formation of disulphide bonds in the nucleus and accessory structures of mammalian spermatozoa during maturation in the epididymis. J. Reprod. Fertil. [Suppl.], 13:65-75. Calvin, H.I., C.C. Yu, and J.M. Bedford (1973) Effects of epididymal maturation, zinc (11)and copper (11) on the reactive sulfhydryl content of structural elements in rat spermatozoa. Exp. Cell Res., 81:333-341. Calvin, H.I., K. Grosshans, and E.J. Blake (1986) Estimation and manipulation of glutathione levels in prepubertal mouse ovaries and ova: Relevance to sperm nucleus transformations in the fertilized egg. Gamete Res., 14:265-275. Caranco, A,, R. Reyes, V.M. Magdaleno, L. Huacuja, 0. Hernandez, A. Rosado, H. Mechant, and N.M. Delgado (1983) Heparin-induced nuclei decondensation of mammalian epididymal spermatozoa. Arch. Androl., 10:213-218. Chargaff, E., and K.B Olson (1938) Studies on the chemistry

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