Changes in sperm nuclei during spermiogenesis and epididymal maturation

CHANGES

IN SPERM NUCLEI AND EPIDIDYMAL M. L. MEISTRICH,’

DURING

SPERMIOGENESIS

MATURATION

B. 0. REID’ and W. J. BARCELLONAZ

‘Section of Experimental Radiotherapy, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 7702.5, and =Deparrment of Biology, Texas Christian University, Fort Worth, TX 76129, USA

SUMMARY Mouse and rat spermatid nuclei pass abruptly through developmental stages characterized by increased resistance to disruption by various agents. The mouse spermatid nucleus becomes resistant to sonication at step 12, resistant to digestion by trypsin-DNase at step 15and resistant to lysis by SDS between the testis and the caput epididymis. These alterations are correlated with changes in the basic nuclear proteins.

The nucleus of the mammalian spermatid undergoes a series of changes in its nucleoprotein composition [l-6] and cytochemistry [7-141 during spermiogenesis and transport from the testis to the epididymis. We report here stepwise changes in the resistance of mouse and rat sperm nuclei to disruption by mechanical and chemical agents and relate these alterations to stepwise changes in the nucleoproteins. MATERIALS

AND METHODS

The mice used were C,HfiBu males between 9 and 15 weeks of age weighing approx. 30 g, and maintained in a specific pathogen-free colony. The rats used were Sprague-Dawley males with body weights between 250 and 300 g. Mouse testis cells were radioactively labelled by intraperitoneal injection of a total of 3 &i/g body weight of methyl-[3H]TdR, spec. act. >l Ci/mM, Schwarz-Mann given in two equal injections spaced 15 h apart. The testes and epididymides were removed immediately after sacrifice of the animals. In one experiment, efferent ducts were separated under a dissecting microscope. The epididymis was cut into several reErptl Cell Res 99 (1976)

gions, identified using the nomenclature of Reid & Cleland [IS] as follows: proximal segment of caput, zone I and proximal part of zone 2; entire caput, zones 14 A; cauda, zones 6 A and 6B. The tunica albuginea was removed from the testes prior to homogenization. Procedures for preparation of trypsin-resistant mouse testicular sperm nuclei were identical to those described previously for epididymal sperm [l6], except as noted below. Two testes were homogenized in distilled water, sonicated for I min at a dial setting of 6 with a Branson W185 sonifier fitted with a micronrobe, and filtered through an 80 urn pore stainless steel screen (200 mesh, W. S. Tyler, Inc., Houston, TX) to remove debris. After removal of an aliquot for determination of total radioactivity in the homogenate, the sperm heads were washed by centtifugation through a layer of bovine serum albumin, resuspended in a solution containing 0.9 mM MgCI,, 1.7% T&on X-100. 0.19% crude trypsin (Trypsin solution. 2.5%. GIBCO) and 15 &ml of crude DNase (bovine pancreatic DNase I, Sigma), and incubated at room temperature (23f 1°C) for 25 mm, unless otherwise indicated. The sperm heads were pelletted, resuspended in distilled water, passed through a 26gauge needle and filtered through a IO pm pore nylon screen (HC-IO Nitex, TET/Kressilk Products, Inc., Houston, TX). Following centrifugation and resuspension in 5 ml distilled water, the samples, containinn 9.3~ l@ merm heads. were filtered onto GFlC filters-for detednation of radioactivity in the sperm heads by liquid scintillation counting. The extensive washing procedure was employed because we were

Changes in spermatid nuclei previously using these preparations to detect small amounts of meiotic DNA synthesis [16]. Sonication-resistant sperm heads were prepared by homogenizing two mouse testes in 5 ml of distilled water, adding 1.2 ml of 5 ‘% Triton X-100, sonicating as described above and filtering through a IO pm screen. A 0.1 ml aliquot was removed and placed in 0.9 ml NCS solubilizer (Amersham Searle) for determination of total radioactivity and the remainder filtered onto Whatman GF/C filters for determination of radioactivity within sperm heads. In addition to the procedure described above for the mouse, the trypsin resistance of mouse and rat sperm heads was assayed in crude sonicated homogenates to which MgCI, (0.9 mM), trypsin (0.21 %) and DNase (I7 yg/ml) were added. To test the effect of reduction of disulfide linkages on trypsin resistance, sperm heads were spun and resuspended in 25 mM Tris - I mM MgCI, buffer (pH 8.0). Then 0. I vol of either plain buffer or buffer containing 100 mM DTT (dithiothreitol. Calbiochem) was added and the sample was incubated at room temperature for 30 min. after which trypsin and DNase were added. Resistance to SDS (sodium dodecyl sulfate) was assayed by mixing equal volumes of homogenates and 2 % SDS (sodium lauryl sulfate. Fisher Scientific) in water (pH 9) at room temperature. All counts of nuclei were performed using a hemacytometer and phase contrast microscope. The procedures for electron microscopy were as described previously [ 161.

73

RESULTS Sonication of rat or mouse testicular homogenates in distilled water disrupts all cells and nuclei, except for the elongated nuclei of late spermatids, which we shall refer to as sperm heads. From each mouse, 3.0~ IO7 testicular sperm heads are obtained. Some of the nuclei appeared to correspond to spermatids as early as late step IO [17]. However. exact staging is not reliable because of swelling of the caudal end of the less mature spermatids in distilled water. These nuclei were then centrifuged and resuspended, but only 2.6~ IO7 could be recovered despite variation of buffer and centrifugation conditions. Measurements of trypsin digestion of sperm heads prior to and after centrifugation indicates that the heads lost upon centrifugation are trypsinsensitive since no loss of trypsin-resistant

Table 1. Resistance properties of testicular and epididymal sperm nucleia Source of sperm heads

Number of sperm heads per animal

% Sonicationresistant

% Trypsinresistant

% Tryspinresistant after DTT treatment

3.0x

IO’

loo

42b. 49c. 57

0”

I .9x IO” 1.5x IO’ 2.5x IO’

100 loo 100

N.D.’ N.D. IOO’d

N.D. N.D. 0”

I (MP 100 IO0

4.4x IO” 3x10”

100 100

6P.64” N.D.

0” N.D.

(I I)’ I O(Y

4.8x IO’ 1.7X10” 3.4x IO”

100 loo 100

N.D. 97”. 97” I ood

0” 0” 0”

100” IO0 100

% SDS resistant __-

,Morr.s C’ Testis Caput epididymidis Proximal segment Entire caput Cauda epididymidis

(2)’

RCll Testis Efferent ducts Caput epididymidis Proximal segment Entire caput Cauda epididymidis

‘I Counts were performed after approx. 25 min of incubation in trypsin-DNase or SDS. h Treated prior to centrifugation step. homogenate in distilled H,O. r Treated after one centrifugation step in MgCI,-Triton solution. Calculated on assumption lost upon centrifugation were trypsin-sensitive. ’ Treated after centrifugation step in Tris-MgCl, buffer. Same assumption as in’. e Markedly swollen after 25 min of incubation; all were lysed within 2 h. ’ Not determined. B Moderately swollen. but none were lysed after 2 h. h Slightly swollen.

that

sperm

heads

14

Meistrich, Reid and Barcellona

IO-

c

Fig. 1. Abscissa: incubation time (min); ordinure: sperm heads/2 testes (X IO-‘). Time course of disappearance of mouse testis sperm heads upon incubation at room temperature with trypsin and DNase (O-O). O-O, Samples in which trypsin and DNase were omitted. DNase alone was also shown to have no effect: Cl. number of heads present after sonication but prior to the first centrifugation. The heads lost during this centrifugation step appear to be trypsin-sensitive. Counts of sperm heads at short incubation times were obtained using soybean trypsin inhibitor (Worthington) to stop the reaction.

heads occurred (table 1). Incubation of either mouse or rat testicular sperm heads with trypsin at room temperature results in a rapid loss of about half of the nuclei with the other half essentially resistant to digestion under these conditions (fig. 1). The “resistant” testicular sperm heads are, however, digested when incubated with trypsin for longer times or at 37°C. In contrast, epididymal sperm heads appear completely resistant to trypsin treatment at room temperature and at 37°C. After prior incubation with DTT, however, both testicular and epididymal sperm heads are rapidly digested by trypsin; the rate of digestion is dependent on the length of DTT preincubation. Testicular and epididymal sperm heads also differ in their sensitivity to SDS. Incubation of testicular homogenates in 1% SDS at room temperature results in immediate swelling and rapid disappearance of all sperm heads. No efferent duct or epiErprl Cc// Re.\ 99 (1976)

didymal sperm heads are lysed by this treatment, even after 5 h incubation [18], although sperm heads from the efferent ducts and from the proximal segment of the caput epididymidis were swollen after 1 h in SDS. Although testicular and epididymal sperm heads subjected to the trypsin preparation procedure appear similar at the light microscopic level, electron micrographs reveal striking differences (fig. 2). Mouse epididymal sperm head chromatin is generally more condensed than that of testicular sperm heads. In some testicular sperm head preparations there are fine fibers extending out peripherally a short distance from the heads, as well as small chromatin aggregates interspersed between sperm heads (fig. 2a). In one preparation, however, these aggregates are in contact with the surface of the testicular sperm heads, rather than being interspersed between them, indicating that the aggregated chromatin is of testicular sperm head origin rather than a contaminant from other cell types. Chromatin aggregates are absent in epididymal sperm head preparations and peripheral chromatin fibers are rarely seen. The only contaminants observed in the testicular preparations are collagen fibrils, presumably from the basement membrane of the seminiferous tubules. During spermatogenesis essentially all DNA synthesis ceases at the end of the premeiotic interphase (preleptotene primary spermatocyte) [16, 19, 201 and the kinetics of spermatogenesis is precisely regulated [20, 211. Therefore, we can calculate the stages at which the sperm nuclei acquire resistance to sonication and then to trypsin-DNase from the times of appearance of radioactivity from injected [“H]TdR in these nuclei. The radioactivity in the mouse sperm head preparations remains very low for several weeks (fig. 3).

Changes

2. Electron micrographs of sperm head preparations. (a) Testicular sperm treated with sonication, trypsin, DNase and Triton X-100 as described in Methods. Aggregations of chromatin material (short arrows) and collagen fibers (long arrows) are indi-

Fig.

in spermatid

nuclei

75

cated. X 10 100; (b) epididymai sperm heads prepared as described previously [16], but without SDS treatment. Arrows indicate residual membrane fragments along the periphery of sperm heads. x 11800. Exptl Cell Res 99 (1976)

76

Meistrich, Reid and Barcellona

resistant heads because fewer sperm are trypsin-resistant, losses are incurred in the washing steps and partial degradation of the sperm heads with the enzymes occurs. The radioactivity in total testicular homogenates, as well as in the sperm head preparations, begins to decline between days 25 and 26. The curves of increase and loss of radioactivity in sperm head preparations (fig. 31, were fit with exponential functions which are a consequence of decreasing incorpora24 26 4 16 20 32 a 12 0 tion into earlier spermatogonial stages. as Fig. S. Abscissa: time after [“H]TdR injection (days); will be described elsewhere [22]. The paraordinare: radioact. (cpmx 1O-4). Radioactivity (per 2 testes) in testis homogenates meters obtained represent the times when (0.. . 0); sonication-resistant sperm head preparations (B- - 4); and trypsin-resistant sperm heads the exponential curves start rising and preparations (O-O), from mice sacrificed at various when they start falling. These values, times after injection of [3H]TdR. The time interval was therefore, correspond to the times when measured from the second of two injections given 15h apart. Each point represents the average of three the cells labelled as preleptotene spermatosamples, each prepared from an individual mouse. cytes begin to acquire resistance and when Symbols with arrows (a) indicate points which are off the scale. Error bars represent S.E. of the 3 measure- these cells leave the testis. These results ments. For total testicular homogenates, error bars are not shown; the average fractional S.E. is 0.10. indicate that labelled cells first acquire The increases and decreases in radioactivity were fit sonication-resistance at 19.6 days, trypsinwith exponential curves by computer [22]. resistance at 22.6 days and leave the testis at 25.5 days after injection. These times Through day 20, less than 10 cpm are correspond to mid-step 12, early step 1.5and present in the extensively washed trypsin- the end of step 16 of spermiogenesis. reresistant preparations and through day 18, spectively. The mouse spermatid nuclei, less than 1000 cpm are present on the filter therefore, are trypsin-resistant for 2.9 days with sperm heads from the sonicated testis and sonication-resistant for 5.9 days. These homogenate. On day 19, a slight but sig- kinetic data indicate that 49 % of the $onicanificant rise in the radioactivity in the tion-resistant heads are also resistant to sonication-resistant testicular sperm heads trypsin, in agreement with results from is observed, and on day 20, the counts rise microscope counts presented in table I. dramatically. Between days 23 and 26, Analogous results obtained with the rat 75% of the total testicular radioactivity is indicate that the onset of sonicationfound in the sonication-resistant sperm resistance occurs at late step 12 [23:]. The times of onset of sonication-reheads. In trypsin-resistant sperm heads, a significant increase in radioactivity is first sistance, trypsin-resistance, and SDS-reobserved on day 21 and becomes most sistance appear to be extremely well lodramatic on day 23. Between days 25 and calized. There are, however, slight but sig30, 30 % of the total testis radioactivity is in nificant increases in radioactivity in the testhe trypsin-resistant preparations. This ticular sperm head preparations at one or radioactivity is lower than with sonication- two days prior to the calculated onset of ExptlCellRes

99 (1976)

Changes in sprrmrrtid

resistance. This could result from extension of DNA synthesis at lower levels into early leptotene [17, 20, 241, dispersion in the kinetics of spermatogenesis, or a gradual onset of the resistance.

nuclei

77

most mammalian species [2], is synthesized and replaces TP and TP2. Little crosslinking of Sl occurs in the testis, but this protein is highly crosslinked in epididymal sperm [4, 6, 271. Autoradiographic data indicate two phases of nucleoprotein synthesis in elongating ram spermatids [28]. The first phase is DISCUSSION characterized by greater incorporation The results presented here show clearly the of lysine and the later phase by greater existence of three transitions of the sperm incorporation of arginine and cysteine. nucleus. Spermatids at step 12 acquire reCorrelating the data on resistance to nusistance to mechanical disruption by sonica- clear disruption with nucleoprotein changtion. At the start of step 1.5in the mouse, es, we conclude that histone-containing spermatids become partially resistant to the chromatin is sensitive to sonication. When action of trypsin. During transport from the histones are replaced by arginine-lysine testis to the caput epididymidis, the sperm rich spermatidal proteins, protamine in become totally trypsin-resistant, as well as the mouse or TP plus TP2 in the rat, the SDS-resistant. chromatin (and the nuclear shape as well) Nucleoprotein synthesis, as indicated by becomes resistant to sonication, although it high levels of arginine incorporation, is is still very sensitive to trypsin. When these most active during steps 11-14 of mouse proteins are subsequently replaced by the spermiogenesis [19, 251. Also, at these sperm basic protein (S 1, in the rat), which stages, the ultrastructure of the chromatin forms some disulfide crosslinks. the chrochanges from a beaded pattern, typical of matin becomes relatively resistant to histones, to a smooth fiber [25]. An ar- trypsin, although it is still sensitive to SDS. ginine-lysine rich nucleoprotein (mouse When more extensive disulfide crosslinking protamine) is synthesized at about step 12 of this protein occurs as sperm leave the [ 11. Although Lam & Bruce claim that this testis and pass through the epididymis [4, protein is also present in epididymal sperm, 18, 271, the chromatin becomes completely other workers have instead found more trypsin-resistant and refractory to SDS. It highly arginine-rich protein(s), also con- is likely that these changes in nucleotaining cysteine, in epididymal sperm [26]. proteins are also responsible for the cytoIf the latter observation is correct, the chemical changes which also occur at these nucleoprotein transitions in the mouse times. would correlate with those in the rat. In the The precise localization of the times of rat, two new basic proteins, TP (equivalent onset of resistance is surprising since major to mouse protamine in amino acid composi- changes of the entire nucleoprotein completion [3, 51 and TP2 [5, 61 are synthesized ment are required. Different regions of the during steps 12-15 and totally replace the nucleus of the elongated spermatid have histones and most of the nonhistones. Dur- been shown to possess different chromatin ing steps 16-18 (corresponding to 14-15 in ultrastructure [25]. However, our data on the mouse), an additional protein Sl [3,23], the onset of sonication and of trypsin-resimilar to the sperm basic proteins from sistance indicate that each replacement of

78

Meistrich, Reid and Barc~rllor~rr

nucleoproteins is localized in the maturation sequence to within one or two days. The onset of SDS-resistance occurs just as the sperm pass from the testis to the efferent ducts, presumably representing the formation of a sufficient number of disulfide bonds necessary to stabilize the sperm head against detergent action. Thus it appears that disulfide bonds rapidly form just as sperm heads leave the testis. Marushige & Dixon [29] reported probable basic nucleoprotein degradation products in mature trout testes and postulated that proteolytic degradation of histones is involved in nucleoprotein replacement during spermiogenesis. Our observation of the differential trypsin sensitivity of chromatin when it contains the arginine-lysine rich spermatidal proteins and when it contains the crosslinked arginine-cysteine rich sperm protein, demonstrates that such degradation of nucleoproteins can be selective. Finally, the differential sensitivity of spermatid nuclei to disruption presents a method for isolation of nuclei of cells in specific stages of spermiogenesis. These methods have been employed in several recent biochemical studies [4, 5, 6, 231.

This work was supported by USPHS grants CA-17364 and CA-06294 from the NCI. a arant from the Ponulation Council, New York, Contract FDA 73-204 and by the Texas Christian University Research Foundation. We thank Drs S. R. Grimes and R. D. Platz for many stimulating discussions. In addition, we are grateful to Calvin Nowack and his staff for the supply and care of the mice used in these experiments.

Exprl

Cd

R<,.\ 99 llY76)

REFERENCES 1. Lam. D M K &Bruce, W R, J cell physio178 (1971) 13. 2. Monfoort, C H, Schiphof, R. Rozijn, T H & Steyn-Parve. E D, Biochim biophys acta 322 (1973) 173. Kistler, W S, Geroch, M E & Williams-Ashman, H G. J biol them 248 (1973) 4532. Marushige, Y & Marushige, K, J biol them 250 (1975) 39. Platz, R D, Grimes, S R, Meistrich, M L & Hnilica. L S. J biol them 250 (1975) 5791. 6 Grimes, S R, Platz, R D,‘Meistrich, M L & Hnilica, L S, Biochem biophys res commun 67 (1975) 182. 7. Monesi, V, Exp cell res 39 (1965) 197. 8. Rinaertz. N R, Gledhill. B L & Dariynkiewicz. Z, Exp cell res 62 ( 1970)204. 9. Gledhill. B L. J renrod fert. SUDDI.I3 (1971) 77 10. Brachet, J, Hulin,‘N & Guermant, J, Exp cell res 51 (1968) 509. I I. Leblond. C P & Clermont, Y, Am j anat 90 (1952) 167. 12. Meistrich, M L, Bruce, W R & Clermont, Y, Exp cell res 79 (1973) 213. 13. Loir, M & Hocherau-de Reviers, M T, J reprod fert 31 (1972) 127. 14. Loir, M, Compt rend acad sci 27 I (1970) 1634. IS. Reid, B L & Cleland, K W, Aust j zool 5 (1957) 221. 16. Meistrich, M L. Reid, B 0 & Barcellona, W J, J cell biol 64 (1975) 2 I I. 17. Oakberg, E F, Am j anat 99 (1956) 391. IS. Calvin, H I & Bedford, J M, J reprod fert, suppl. I3 (1971) 65. 19. Monesi, V. J cell biol I4 (1962) I. 20. Clermont, Y & Trott, M, Fertil steril 5 (1969) 805. 21. Oakberg, E F. Am j anat 99 (1956) 507. 22 Meistrich. M L. in preparation. 23 Grimes. S R, Platz, R D, Meistrich. M L & Hnilica. L S. In preparation. 24. Huckins, C. Personal communication. 25. Kierszenbaum, A L & Tres, L L, J cell biol 65 (1975) 258. 26 Bellve, A R & Romrell. L J, J cell biol 63 (1974) l9a. 27 Calvin, H I, Yu, C C & Bedford, J M, Exp cell res 81 (1973) 333. 28 Loir, M, Ann biol anim biochem biophys I2 (1972) 411. 29 Marushige, K & Dixon, G H. J biol them 246 ( 197I) 5799. Received July 2, 1975 Accepted September 29, 1975