Changes in deoxyribonucleoprotein during spermiogenesis in the bull

Experimental Cell Research62 (1970) 204-218

CHANGES

IN DEOXYRIBONUCLEOPROTEIN

DURING

SPERMIOGENESIS

IN THE BULL

Sensitivity of DNA to Heat Denaturation NILS R. RINGERTZ,

BARTON L. GLEDHILL

and ZBIGNIEW

DAR5?YNKIEWICZ1

Institute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, Sweden, Institute for Obstetrics and Gynecology, Veteriniirhiigskolan, Stockholm, Sweden, and Section of Reproduction, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, Pa 19348, USA

SUMMARY The thermal stability of DNA at various stages of spermiogenesis in bulls was studied by new cytochemical techniques. Fixed smears of testicular cells were heated to various temperatures in the presence of formaldehyde. After cooling, the extent of single-stranded DNA regions was measured by microspectrophotometric determination of the increment in UV-absorbancy at 265 nm or by microfluorimetry after acridine orange (AO) staining. The extent to which DNA had become denatured into a single-stranded state was determined from the magnitude of a shift in the fluorescence emission spectrum. Although UV-microsoectronhotometrv and acridine oranne microfluorimetry measure -different physical phenomena; the present results show that it is PO;sible to follow the denaturation of DNA (“melting urofile”) in intact cell nuclei bv both these techniques. Since both techniques were used in parallel throughout the present investigation it has been possible to compare the two techniques relative to each other. The main biological conclusion from the present work is that duringspermiogenesis theDNA becomes progressively more resistant to heat denaturation. This is thought to be due to changes in the state and composition of the deoxyribonucleoprotein. The relationship of these DNP changes to genome inactivation and chromatin condensation is briefly discussed.

Differentiation of spermatids into spermatozoa involves a condensation of nuclear chromatin with a change in nuclear morphology. Concurrent with condensation of the chromatin there are changes in the composition and cytochemical properties of the deoxyribonucleoprotein (DNP) complex. In fish, somatic histones are known to be replaced by arginine-rich protamines [ 14, 151. Though biochemical work has failed to identify protamines in mammalian spermatozoa1 heads, IPresent address: Boston Biomedical Research Institute, Department of Connective Tissue Research, 20 Stamford Street, Boston, Mass. 02114, USA. Exptl Cell Res 62

the alteration in cytochemical properties of the DNP which occurs during spermiogenesis in the bull [12], strongly suggests that changesin the composition and physical properties of the DNP similar to those found in fish also occur during the chromatin condensation typical of mammalian spermiogenesis. These changes in the DNP express themselves as a decrease in stainability by the Feulgen reaction, as a lowered capacity to bind basic dyes, as an increase in proteinbound arginine groups, as an increasein the total number of dye-binding basic groups in the protein component of the DNP complex

Changes in DNP during bovine spermiogenesis

[12], and as a decrease in the ability of the complex to bind SH-AMD [9]. The DNP changes observed during spermiogenesis are of general biological interest since they are accompanied by a complete inactivation of the genome with respect to RNA synthesis and also by marked changes in the state of condensation of the chromatin. Spermiogenesis can in certain respects be considered a model systemin which it may be possible to examine the molecular changes underlying genome inactivation and chromatin condensation. Some newly developed techniques for studying the thermal denaturation properties of DNA in intact cell nuclei [20] have made possible, the further analysis of DNP changes during spermiogenesis.The sensitivity of DNA to heat denaturation is dependent upon the extent and mode of protein binding to DNA in the DNP complex. The aim of the present investigation has been to examine the sensitivity of DNA to heat denaturation at various stages of spermiogenesis in order to elucidate the previously observed [12] changes in DNAprotein interaction. MATERIALS AND METHODS

ing the smears in a 0.15 M NaCl, 0.015 M Na-citrate (SW) solution containing 4% formaldehyde (w/v, pH 7.0) for 20 min. After heating, the smears were rapidly cooled in ice-cold SSC. The cells were then dehydrated by passing the smears through a series of ethanol baths whose concentrations increased stepwise until absolute ethanol was reached. Quartz slides were transferred to glycerol and allowed to equilibrate with this medium for 24 h before application of quartz coverslips. The cells smeared on quartz slides were then analyzed by scanning and integrating UV microspectrophotometry in an instrument developed by Caspersson & Lomakka [5]. UV absorption measurements at 265 and 315 nm are reported as total extinction (Ese5and E,& integrated over the area of the nucleus (E =@). Smears which had been made on glass Btirkerhaemacytometer slides were also subjected to the denaturation procedure as outlined above. The cells were then rehydrated in a series of ethanol baths of decreasing concentrations. Following this, the slides were stained with acridine orange (AO). Details of both the heating and staining procedures have been described in a nrevious communication 1201.Microfluorimetry of *AO-stained cells was done with a Zeiss microfluorimeter (for details. see reference 1221). The fluorescence intensities are reported in arb&G units. Since the instrument used in the present investigation has interference filters for the selection of wavelengths of the emitted light and not a monochromator as in a previously published work [12], the FS80/F630 ratios (alpha; rx) differ somewhat from those reported earlier. The cr-values in the present report can be compared with previous data [12] if multiplied by a factor of 1.3.

Chemical methods DNA was isolated from calf thymus by the method of Chareaff 17l. The heat denaturation nronerties of this DNA were studied by heating 3 ml samples of DNA (A,.. = 0.3) for 20 min in the 4 % formaldehvdeSSC solution used for the cytochemical experiments. After rapid cooling, the optical densities of the solutions were recorded at 265 and 315 nm in a Zeiss PMQII spectrophotometer. The optical densities recorded at 315 nm were insignificant and therefore no corrections for non-specific light losses were made. \

Specimen preparation Seven bulls of the Swedish Red and White breed were used for this investigation. Three of these bulls were of normal fertility (one of them did have abnormally high percentages of malformed cells on the testicle scrape preparations) whereas four showed reduced fertility. Complete clinical data on these bulls will be presented elsewhere (Gledhill, Ringertz & Dariynkiewicz. To be published). Cells were collected by scraping freshly cut surfaces of testicles within a few minutes of castration, and by collecting ejaculates with an artificial vagina just prior to castration. The cells were washed in buffered balanced salt solution [17], smeared onto slides and fixed in ethanol : acetone (l:l, v/v) as described in an earlier publication [12]. RNase treatments were also performed as previously described.

Cytochemical methods After stepwise hydration of the fixed cells the heat denaturation experiments were performed by heat-

205

_“I

Identification

of cell stages

The present investigation has been restricted to cells representative of four phases of male germ cell differentiation: round spermatids, elongated spermatids, testicular spermatozoa and ejaculated spermatozoa (fig. 1). The recognition of these cell types was based on their morphology in the phase-contrast, UV and fluorescence microscopes. Though the distinction of these cell types did not present a problem, it is clear that the point of differentiation between a round and an elongated spermatid is less precise than is the identification of spermatozoa. Elongated spermatids have been taken as any stage of development between round spermatids and cells clearly Exptl Cell Res 62

206

N. R. Ringertz et al.

Fig. I. Schematic drawings illustrating the morphology of the four different types of cells investigated. showing the morphological characteristics of spermatozoa. Thus, elongated includes elongating spermatids. For further details on the morphological guidelines used to identify cell types, several reviews, e.g. references [2, 231, can be consulted.

spermatids as well as for testicular and ejaculated spermatozoa. Nevertheless, the data given in figs 2-5, 9 and 10 and in tables 1 and 3 have been corrected for non-specific light losses by subtraction of the extinction recorded at 315 nm. RESULTS The shape of the UV absorption spectra Factors influencing UV absorbance of varied between the different stages of germ spermatid nuclei and sperm heads cell maturation. Pretreatment of the cells also The results obtained with scanning and inte- affected the shape of the curves. As can be grating UV microspectrophotometry of indi- seenin fig. 2, round spermatids show a greater vidual spermatid nuclei and sperm heads are amount of absorption between 267 and 280 summarized in table 1. Round spermatids nm than do cells at later stagesof maturation. were found to have a greater amount of nu- The formaldehyde treatment used in the heat clear UV absorption at 265 nm than did nu- denaturation procedure was found to increase clei in cells from later stagesof spermiogene- the UV absorption of round spermatids in sis. As the cells became increasingly mature, the range of 270 to 300 nm, the extinction the amount of UV absorption decreased. value at 300 nm being doubled by the formalRNase treatment reduced the UV absorption dehyde treatment. However, the absorption of round spermatid nuclei, but had little at 265 nm, i.e. the wavelength usedfor absorpeffect on cells representing later stages of tion measurements in the heat denaturation spermiogenesis.For the most part, the higher experiments, was not significantly altered by UV absorption of round spermatids therefore the formaldehyde treatment. seemedto be due to RNA which was removable by RNase digestion. Another significant Effect of heat denaturation on nuclear UV reason for the higher absorption of round absorption spermatids relative to other types of haploid The UV spectral changes induced by heating cells was the greater amount of light scat- cells to 100°C are illustrated in fig. 2. An tering caused by this type of cell. The light increase in absorption occurred throughout losses due to scattering (estimated as E& the region of 250 to 280 nm. At 240 and 300 were found to be insignificant for elongated nm the increase was negligible for elongated Exptl Cell Res 62

Changes in DNP during bovine spermiogenesis

207

Table 1. The effect of RNase treatment and heating to 100°C on the total extinction at245 nm (E,,,) of spermatids and spermatozoa

Cell type Round spermatids uncorrectedb corrected Elongated spermatids uncorrected corrected Testicular sperm uncorrected corrected Ejaculated sperm uncorrected corrected

22°C Undigested=

22°C RNase digested

100°C RNase digested

% increase in Eze5 22”c-1oo”c (RNase digested)

12.9 +0.7 12.5

8.0 7.6

11.4 10.9

43

7.6kO.l 7.4

7.8 7.5

9.5 9.2

23

6.7kO.l 6.6

7.0 6.8

9.1 8.9

31

6.6kO.l 6.5

2:::

8.9 8.9

33

a Cells were fixed in ethanol-acetone (1:l) and then without RNase and cold TCA exposure, were placed for 20 min at 22°C in the SSC-formaldehyde solution used in the heat denaturation experiments. Another set of slides was exposed to RNase digestion followed by extraction with cold TCA as indicated in the material and methods section. Data on undigested cells are based on one normally fertile bull. Data on RNase treated cells are based on seven animals (See fig. 3.) ’ Non-specific light losses were estimated at 315 nm. Corrections were performed by subtracting Ea15from E,,,. All values are given as mean in p2 per head + S.E.M. (n = 10).

spermatids as well as for testicular and ejaculated spermatozoa. The increase in absorbance by elongated spermatids for the region between 265 and 280 nm in the experiment displayed in fig. 2 was lower than usual (compare with fig. 3) for unknown reasons. Figs 3-5 illustrate the change in E,,, associated with increasing temperature. In fig. 3 the corrected E,,, values for each type of cell and for individual animals are given for each temperature in order to illustrate the total experimental and biological variation encountered. The standard error of the mean for each determination varied little. Based on the data summarized in fig. 3 and table 1, it seems that the major source of variation was from one experiment (one animal) to another and that intercellular variation in a single population of cells ,was comparatively small. From the data presented in fig. 3 it is clear

that no major differences existed between the three bulls of normal fertility and the four bulls of reduced fertility with regard to the increased 265 nm absorption resultant from increased temperature. It should be emphasized at this point that, in both fertile and infertile animals, measurementswere restricted to cells with a normal morphological appearance. In smears made from semen and testicular scrapes from the infertile animals, numerous types of abnormalities were observed. The particular cytochemical properties of these cells as well as a correlation with the clinical data will be discussedin a separatepublication [l 11. Since no differences were found between morphologically normal cells from the fertile and infertile bulls, E,,, mean values as indicated in figs 3, 4 were calculated by pooling data from all seven animals. In all experiments and for all cell types, a marked increase in nuclear EZa6was observed Exptl Cell Res 62

208 N. R. Ringertz et al. E 265

ELONGATED

ROUND SPERMATIDS

SPERMATIDS

1L.O

12.0

10.0

E.0

6.0

40

2.0

EJACULATED

TESTICULAR SPERMATOZOA

2LO

250

260

270

280

290

300

2LO

250

260

270

280

290

300

Fig. 2. Abscissa: wavelength, nm; ordinate: absorbancy. UV absorption spectra of spermatids and spermatozoa from one fertile bull. Cells fixed in ethanol-acetone ( x - x ), ethanol-acetone fixation and formaldehyde treatment at 22°C ( A . + . A), ethanol-acetone fixation and heating to 100°C in the presence of formaldehyde (o-o).

when the cells were heated to temperatures of between 60°C and 100°C. Some increase occurred in round spermatids between 22°C and 60°C which was followed by the major increase between 60°C and 75°C. A levelling off occurred between 75°C and 95°C. The data also indicate that some increase occurs between 95°C and 100°C although a considerable variation exists between different animals. The increase in Ezasfor elongated spermatids, testicular and ejaculated spermatozoa began at a temperature which was higher than that seen for the initial increase of the round spermatids (figs 4, 5). Although the total extinction values have Exptl Cell Res 62

been corrected for light scatter by subtraction of the respective E,,, values, the starting (22°C) and ending (100°C) points differ for the four cell types examined. Round and elongated spermatids have a higher E,,, at 22°C than do testicular and ejaculated spermatozoa. At 100°C the elongated spermatids and both types of spermatozoa show approximately the same Easswhereas the value for round spermatids at this temperature is roughly 20 Y! higher. Therefore, it is meaningful to express the results not only as changes in total Et85but also in terms of relative (%) increase. As shown in fig. 5, the percent increasein E,,, between 22°C and 100°C is the

Changesin DNP during bovine spermiogenesis 209 49-121169

ELONGATED SPEWATIDS

TESTICULAR SPERMATOZOA

EJACULATED SPERMATOZOA

Fig. 3. Abscissa: Ees5;ordinate: temperature, “C. Increase in nuclear Ese6induced by heating smears of testicular cells and ejaculated spermatozoa in SSC solution containing 4 % formaldehyde. These microspectrophotometric data are derived from 3 bulls of normal fertility (0) and 4 bulls of reduced fertility ( x ). One of the bulls with normal fertility showed excessive numbers of head and tail abnormalities on testicle scrape preparations (0). The measurements have, however, been restricted to cells with normal morphology as indicated in fig. 1. No difference in the Eaa5values was noted in morphologically normal cells obtained from either fertile or infertile bulls.

samefor round spermatids, testicular and ejac- curred since testicular spermatozoa are more ulated spermatozoa, whereas the correspond- sensitive to heat denaturation than are ejacing increase for elongated spermatids is small- ulated spermatozoa (fig. 5). These cytochemical melting profiles show er. In fig. 5 it can be seen that the four cell types have a different temperature region for a more gradual increase in E,,, than is usually . . observed in standard, biochemical heat denatheir mam increase in E,,,. A correlation exists between stage of cellular maturation turation experiments with DNA or DNP. and the sensitivity of DNA to heat denatura- This is not necessarily due to the presence of tion, ejaculated spermatozoa being most re- formaldehyde in the cytochemical heat denasistant and round spermatids most sensitive turation procedure since purified calf-thymus to the effects of heat denaturation. Thus, DNA heated in SSC containing 4% formalduring spermiogenesis in the bull, DNA be- dehyde gave a very sharp increase in UV abcomes progressively more stabilized against sorption (fig. 10). A comparison of the bioheat denaturation. This process continues chemical and cytochemical melting profiles after the main morphological changeshave oc- also reveals that, with the exception of round 14-

701817

Exptl Cell Res 62

210

N. R. Ringertz et al.

I

, 20

60

70

80

90

MO

Fig. 4. Abscissa: Eaes;ordinate: temperature, “C. Increase in mean nuclear E,,, of round (O-O) and elongated ( x - x ) spermatids, testicular (A-.-A) and ejaculated ( 0 * . . 0) spermatozoa. The curves present mean values derived from the pooled data from the 7 bulls referred to in fig. 3.

spermatids, the total increase in Eza5is somewhat smaller in the cytochemical experiments than in the biochemical experiments.

the marked increase in F,,, after heating to 100°C (table 2). The extent to which heating induced an increase in F,,,/F,, ratios (LX)varied somewhat from one experiment (one animal) to another as well as from cell type to cell type. In fig. 6 the individual a-values for each animal and temperature are given to illustrate the variation between experiments. Again, no clear-cut difference existed between the fertile and infertile animals. Although morphologically normal cells from one of the fertile bulls (identified with symbol 0 in fig. 6) gave unusually high ccvalues at 22°C for all stages of maturity, there was no consistent difference between fertile and infertile animals when they were arranged into groups. This aspect will be presented in more detail elsewhere where more attention will be focused on the clinical data and to cytochemical data obtained by other techniques. In fig. 7 the mean a values for each type of cell (pooled data) have been recalculated as

A0 binding of formaldehyde treated and heat denatured nuclei

The A0 binding properties of the different types of cells examined are presented in table 2. In agreement with our previous observations [12], the amount of A0 bound to DNA progressively decreases as the cells become more mature. Concurrently, there is a “red shift” in the fluorescence emission spectrum, i.e. an increase in the F,JF,,, ratio (a). - When the cells are heatedand then stained with A0 the heating induces an increase in the F,,,,/F,, ratio for all the cell types. This red shift is interpreted as reflecting a change in the mode in which DNA binds A0 due to a transition from the doublestranded to the single-stranded state [20]. In addition, there is also a marked increase in the number of A0 molecules bound by testicular and ejaculated spematozoaas shown by Exptl Cell Res 62

l&O-

no-

120.

llO-

looi

io

so70so90

loo

Fig. 5. Abscissa: relative value for Ezas; ordinate: temperature, “C. Percent increase in nuclear 265 nm absorption relative to the starting be6 value (22°C). Symbols are as in fig. 4.

Changes in DNP during bovine spermiogenesis

Table 2. Binding of Acridine

211

Orange to bull spermatids and spermatozoa; FSw valuesa,

a valuesb Characteristic FG8,,at 22°C F,,, at 100°C AFsSO22” to 100~~ (%) a at 22°C a at 100°C Aa 22” to 100°C (%)

Round spermatids 9.8 10.9 11 0.14 0.28 loo

Elongated spermatids 5.3 5.3 0 0.15 0.26 73

Testicular Sperm 2.1 4.1 95 0.18 0.28 56

Ejaculated sperm 1.0 2.9 190 0.19 0.28 47

0 FSeOis used as a rough measure of the total number of A0 molecules bound per nucleus [19] and is presented here in arbitrary units. b CC = F600/F630.It indicated the “redness” of the emitted A0 fluorescence and is related to the proportion of single-stranded to double-stranded DNA nresent. Further, a is independent of the quantity of DNA present [19; 203.

relative values (values at 22°C = 100) in order to compare the per cent increase in a for the individual cell types. Such a comparison shows that the temperature at which the main increase in a occurs is related to the maturity of the cell. As the cell matures the DNA becomes progressively more resistant to heat denaturation as indicated by the increased a values. It appears from fig. 7 that the main increase in thermal stability occurs when the cell is in the epididymis, that is, somewherein time between testicular and ejaculated spermatozoa. This impression is erroneous partly becauseof different starting values. As shown in table 2, ejaculated spematozoa have a higher initial a value; when the cells are heated a decreasein ccactually occurs before the main increase occurs between 75°C and 95°C (figs 6,7). Thus, by choosing the a value at 22°C as a basis for calculation of relative values (fig. 7), the difference in melting profile between testicular and ejaculated spermatozoa becomes greater than it would have been had 60°C or 70°C been chosen as a basis for the comparison. Differences between stages of cellular maturity were also observed when the total amount of A0 binding as reflected in the F,,, values was plotted against temperature (fig.

8). Heating to 100°C markedly increased the F,, values of testicular and ejaculated spcrmatozoa. Changes in FboOwere also noted for round and elongated spermatids. Curves representing these changes, however, had a very complex shape (fig. 8) showing an early increaseand then a decrease. Loss of DNA in the A0 staining procedure or incomplete denaturation

The melting profiles obtained for cells representative of different stagesof spermiogenesis and sperm maturation differ considerably from those of rapidly proliferating cells [3, 131.The fluorescence levels (F,,, and F6& are lower and the increases in red fluorescence (E) are less marked. This situation might result: (i) if the denaturation of DNA were incomplete, (ii) if DNA were lost during the heating and staining procedure, or (iii) if the A0 and UV methods measured different physical phenomena. Since the UV extinction values obtained from heated sperm heads agreed with the values which were expected if all the DNA were still present, it appeared that the A0 melting profiles were not seriously affected by losses of DNA during the heating procedure (which is the same for the two methods). In order to test if DNA losses Exptl Cell Res 62

212 N. R. Ringertz et al. x

Oh5.

ROUND SPERMATIDS Xa

a

_ ELONGATED SPERMATIDS

x

0.35

oJ5

TESTICULAR SPERMATOZOA 0

*

x

EJACULATED SPERMATOZOA

x*

0

OOx DI

OX-

m

60

70

no 90 m

20

50

m

w 30 m

Fig. 6. Abscissa: F59JF6S0(01);ordinate: temperature, “C. Effect of heating on the F600/F5S0 ratio (a) of AO-stained smears of testicular and ejaculated cells. The increase in a reflects the change from a doublsstranded to a single-stranded state of the nuclear DNA. Symbols are used in the same manner as in fig. 3.

occurred when the heated preparations were acetylated and stained according to the A0 procedure [19], ejaculated spermatozoa on several preparations from one bull were measured in the UV microspectrophotometer immediately after heating and then again after the slides were brought through the whole staining procedure except the dye bath. The results presented in table 3 indicate that no major loss of DNA occurs during the acetylation and differentiation steps. Therefore, the shape of the A0 melting profiles is unlikely to be seriously affected by DNA losses during the staining procedure. The maximum increase in Es6s usually amounted to approx. 35 % and is only slightly below the maximum increase observed when isolated bovine DNA was heated in the same solutions (fig. 10). Therefore, the denaturation process appears to be almost complete. Exptl Cell Res 62

DISCUSSION Analysis of the sensitivity of DNA to heat denaturation is a useful biophysical method whereby the base composition of unknown DNA preparations can be determined and the interaction between DNA and various low and high molecular weight cations can be studied. In the present investigation, newly developed cytochemical methods [20] have been used to study changes in the thermal stability of DNA from selected stages of spermiogenesisand sperm maturation in the bull. The transition of DNA from a doublestranded to a single-stranded state has been examined by heating smearsof representative cell populations to various temperatures in the presence of formaldehyde. Under the stated experimental conditions, denaturation of DNA took place but renaturation was prevented by the formaldehyde which reacted

Changes in DNP during bovine spermiogenesis

20

El

70 80 90

100

Fig. 7. Abscissa: F&FS3,, (a); ordinate: temperature, 01, L. Mean values for the relative increase in a (22”C= 100). The four types of cells differ from each other with respect to their initial a-value as indicated in table 2. Eiaculated spermatozoa show a decrease in a between- 22’C and-75°C before the main increase in a occurs. The curves indicate a progressive stabilization of DNA to heat denaturation during spermiogenesis. Symbols are used in the same manner as in fig. 4.

with the amino groups of nucleotide bases. This reaction prevented restoration of hydrogen bonding between base pairs as the preparations returned to room temperature. Thereafter, estimation of the extent to which DNA became single-stranded (“melted”) was determined either by measuring the increase in nuclear EZe5with a scanning and integrating UV microspectrophotometer or by staining the cells with acridine orange (AO) and then analysing the properties of the fluorescence emission spectrum. The latter technique is based on the fact that A0 binds in a monomer form to double-stranded DNA giving rise to a green fluorescence, whereas single-stranded, “denatured” DNA binds the dye in an aggregated form which gives rise to a red fluorescence. By measuring the ratio between the amount of red and green fluorescence emitted (F6BO/F630 = LX),it is possible to follow changes

213

in the proportion of single- to double-stranded regions induced by heating. The results obtained with the W method demonstrate that, in cells which contain negligible amounts of RNA, it is possible to study the thermal stability of DNA in individual cells by determining the effect of heating on the extinction at 265 nm. However, the task of analyzing the kinetics of a 40 % increase in E,,, at the level of the individual nucleus requires the aid of highly precise UV microspectrophotometers such as those developed by Caspersson& Lomakka [5] and used in this investigation. In the present study, all cells were digested with RNase although it was actually only necessaryto remove RNA from the round spermatids (table 1). The absence of measurable amounts of RNase digestible UV absorbing material in the other cell types and the small quantity of RNA

e 0

Fig. 8. Abscissa: FSsO,relative value; ordinate: temperature, “C. Effect of heat denaturation on total A0 bindine capacity of nuclear DNP as reflected by Fee,,.Hea& results in a progressive increase in number of dyebinding sites in the DNP of ejaculated ( q * * . q ) and testicular ( A-*- A) spermatozoa. Round (O-O) and elongated ( A - A) spermatids show an initial increase in F680with maxima at 60°C and 80°C respectively, followed by a decrease. Although difficult to interpret, these curves provide additional support for the evidence suggesting marked changes in the conformation of DNP during spermiogenesis. Exptl Cell Res 62

214

N. R. Ringertz et al.

Fig. 9. Abscissa: Eaas, relative value; ordinate: a, relative value. Correlation between the increase in the nuclear Ezas and the increase in the nuclear a value when testicular and ejaculated cells are subjected to heat denaturation. During the first part of the denaturation process the increase in a appears to be directly proportional to the increase in UV absorption. During the last part of the denaturation process a increases more rapidly than does UV absorbance. The shape of the curve is in approximate agreement with theoretical predictions [20].

present in round spermatids presents a very favorable situation for UV microspectrophotometry. An additional advantage found in this cell system which generally does not occur in other cell systems is that all cells contain the same (haploid) amount of DNA. Intercellular variation, therefore, is reduced to a minimum. All extinction values reported herein have been corrected for non-specific light losses by subtracting the extinction at 315 nm from the ESG5 values. This correction is necessary for only the round spermatids but it has been performed on all data presented in the figures and the tables. The results obtained with A0 microfluorimetry confirm and extend our previous observations [12] that the capacity of DNP to bind A0 progressively decreasesduring spermiogenesis. Heating the cells produces at least two different effects on nuclear A0 binding. At temperatures where the DNA can be expected to melt, A0 fluorescence becomes more red. This phenomenon is shown by the progressive increase in c(. For the four types of male germ cells examined, an a value of 0.28 appears to be the maximum value obtained at 100°C. Based on cytochemical model experiments [13] and our previous experience with other cell-types [3,20,21], this increase in tc indicates a change from the double-stranded to the single-stranded state Exptl Cell Res 62

of the DNA. Although the UV absorption measurementssuggestthat denaturation of the DNA is almost complete at lOO”C, the increase in a is much less than expected from theoretical considerations and previous methodological experiments [20]. The explanation for this discrepancy is not known at present. It is likely, however, that the small increasein ccis associatedwith the fact that the cells examined bind very little A0 in comparison to actively proliferating cells. In spite of this discrepancy it seemscertain that the increase in ccis related to the denaturation process as shown by the increase in UV absorption. If the increase in UV absorption is plotted against the increase in a, the positive relationship illustrated by fig. 9 becomes evident. Up to the point where the denaturaTable 3. Effect of the A0 staining procedure on nuclear UV absorption of jormaldehydetreated and heated ejaculated spermatozoa

4 % formaldehyde treatment with heating to

Before A0 staining procedure E 21xa

22°C 6.6kO.l 95°C 9.4fO.l % increase in EBB6 between 22°C and 95°C 41.5 %

a Meanin paper head rf:S.E.M. (n = 10).

After A0 procedure except dye bath Ezesa 6.7 +O.l 9.2f0.2 37.2 %

Changes in DNP during bovine spermiogenesis

*--*-*-t--

140-

X' :

7

20

Al

q

60 70 80 90 im

Fig. 10. Abscissa: E,,, (curves B, C, D) and absorbancy at 265 nm (curve A); ordinate: temperature, “C. Increase in relative E,,, for DNA extracted from calf-thymus and heated according to the cytochemical procedure (A), round spermatids (bull) (B), hen erythrocyte nuclei (C) and ejaculated bull spermatozoa (0). The DNA of intact cell nuclei “melts” at higher temneratures than does calf thvmus DNA. DNA nresent ;n cell nuclei undergoes a more gradual denaturation than does nurified DNA in solution. The denaturation process appears to begin at 55°C for calf thymus DNA and hen erythrocytes, whereas for ejaculated spermatozoa denaturation begins at 75°C. The shape.of the curve obtained for round spermatids also suggests that the denaturation process begins at 55°C but due to an insufficient number of experimental points this curve has not been plotted between 20°C and 60°C.

tion process is approx. 75 % complete, as judged by the UV method, the increase in a is linearly related to the increase in UV absorption. Beyond this point, however, the relative increase in b: is greater than is the increase in UV, causing the curve to deviate from linearity. The shape of the curve which exemplifies this apparent correlation agrees, in principle, with the predicted correlation [20] between an increase in ccand an increase in the amount of single-stranded regions in the DNA mass. Another phenomenon which was observed was a change in the total number of A0 molecules bound to DNA. During the denatura-

215

tion process, testicular and ejaculated spermatozoa exhibit a marked increasein the total number of A0 molecules bound to their DNA with the highest values being reached at 100°C. Round and elongated spermatids at first show an increase in LXand subsequently show a decreaseso that if F,,, at 22°C is compared with Fssoat 100°C there is little difference in the total number of A0 molecules bound by the DNA. It is likely that these changes in total A0 binding are due to conformational changes in the DNP during heat denaturation. Apart from the observation that denaturation of spermatozoa is associated with an unmasking of dye-binding sites, these data are difficult to interpret. They do demonstrate, however, that certain properties of the DNP complexes change markedly at different levels of germ cell maturity, and that there is a correlation between the stage of maturity and the appearance of the F,,, curve. The melting profiles obtained with cytochemical methods differ from those obtained with biochemical techniques in that the denaturation process of DNA or DNP is extended over a broader temperature range by the former method. This difference could be due to the fact that formaldehyde is used in the cytochemical procedure. However, in the experiments where purified calf-thymus DNA was heated in the sameformaldehyde containing salt solution as used in the cytochemical experiments, the rise in UV absorption was as sharp as in experiments where DNA was denatured by conventional techniques. Thus, formaldehyde does not inhibit the denaturation of pure DNA. At present it is impossible to exclude the possibility that the situation with DNP may be different. Formaldehyde may cause cross-linkage between the various protein groups in the DNP and thereby interfere with the denaturation process. One indication that this may have occurred is the Exptl Cell Res 62

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fact that the maximal increase in EZe5of round spermatids, testicular and ejaculated spermatozoa was 36 to 37 % (elongated spermatids increased by only 30 %) whereas calf thymus DNA showed a 42% increase in E,,, (fig. 10). There are other possible explanations for this discrepancy. One is that the DNA in an intact cell nucleus may have a single-stranded component, whereas chemically isolated calf thymus DNA may be completely double-stranded. Formation of the DNA-histone complex could cause a distortion in the base stacking interaction in the direction of melting. It is quite plausible that changes in the binding of proteins to DNA which occur during spermiogenesis [8, 9, 12, 14, 151affect not only the extent to which the UV absorption increases upon complete heat denaturation but also the extinction coefficient of DNA in the intact cell nucleus. Lindstrom et al. [16] have shown that the extinction coefficient of DNA is markedly dependent upon concentration. The types of cells examined in the present investigation differ from each other with respect to the intranuclear concentration and packing of DNA. Variation in the DNA extinction coefficients therefore may explain not only the differences in UV hyperchromicity, but also the fact that even after removal of most of the RNA and correction for non-specific light losses,the initial E,,, values differ slightly for the different cell types. It is interesting to compare the melting profiles we obtained for spermatids and spermatozoa with the melting profiles obtained by other investigators and for other cell types. The only other attempt to obtain a cytochemical melting profile on intact cells that we are aware of was reported by Chamberlain & Walker [6] for boar spermatozoa. Their experiments were performed using a different approach which was based on the continuous Exptl Cell Res 62

heating and recording of UV absorbancy of single cells in another medium. Therefore, detailed comparisons with the present results cannot be made. However, the extent to which an increased UV absorbancy can be induced is similar for both studies. We failed to detect the absorption “hump” detected by Chamberlain & Walker at 50°C to 60°C. In fig. 10 the melting profiles for round spermatids and ejaculated spermatozoa are compared to the melting profile obtained from hen erythrocyte nuclei [21]. This comparison clearly shows that these three cell types differ considerably from each other with respect to the shape of their UV melting profile and that the hen erythrocyte melting profile is intermediate to those for round spermatids and ejaculated spermatozoa. The A0 melting profiles obtained in the present experiments differ from those observed for HeLa cells, lymphocytes, and hen erythrocytes [20, 211. The main difference is the extent of the increase in cc induced by heating. There are also differences in the shapes of the A0 melting profiles. The significance of these specific differences between cell types as they relate to spermiogenesisand maturation of spermatozoa will be discussed elsewhere. Their cytochemical significance has been presented above. The biological significance attached to the data as an entity is that condensation of nuclear chromatin during spermiogenesisin the bull is associated with a progressive increase in the stability of DNA to heat denaturation. Increased stabilization is observed as round spermatids differentiate into elongated spermatids. It is during this period that the nucleus begins to flatten and the chromatin begins to condense. Further stabilization of DNA is found as elongated spermatids are transformed into testicular spermatozoa, a period when condensation of the chromatin is nearly completed. Of particular interest is the

Changes in DNP during bovine spermiogenesis

difference in thermal stability detected between the DNA of testicular and ejaculated spermatozoa. This difference suggests that the DNA of the spermatozoa1head undergoes further molecular rearrangement during epididymal passage. The reasons for the increased heat stability of DNA during spermiogenesis are difficult to analyse. From the classic biochemical work of Kossel [14, 151we know that the histones characteristic of somatic tissue nuclei are replaced by arginine-rich protamines during spermiogenesis in fish. It is also known that, in several mammalian species, the proteins linked to DNA found in spermatozoa1heads differ from those found in nuclei of somatic tissues (see ref. in [8, lo]). Although biochemical work on bull spermatozoahasfailed to identify protamines, the proteins bound to DNA in the spermatozoa1 heads appear to differ considerably from calf-thymus histones in their amino acid composition [4]. Previously [12], we demonstrated in the bull that during spermiogenesisthe stainability of histones and the arginine content of nuclear proteins markedly increase. These data demonstrate a qualitative change in the type of basic protein present. Concurrently, the number of nuclear binding sites for basic dyes (acridine orange and methyl green) decreased and the reactivity of DNA to the Feulgen reaction changed. After fitting all the results together, we suggestedthat the negatively charged phosphate groups of the DNA were masked by new types of proteins which contained more arginine and were more basic than those proteins typically bound to DNA in somatic cells. The current observation of a progressive stabilization of DNA to heat denaturation during spermiogenesis gives further and stronger support for this suggestion. One consequence of the increasing neutralization of the DNA-phosphate groups is that the DNP

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molecules would become less rigid, attract fewer water molecules, and pack together into a smaller volume. These modifications in DNP observed during spermiogenesisseem to constitute a logical biochemical background for explaining at least part of the mechanism underlying the typical condensation of nuclear chromatin which occurs during mammalian spermiogenesis. RNA synthesis by spermatids is completely stopped during the latter portion of spermiogenesis [18]. It is possible that the modifications to the nuclear DNP described above and/or the marked condensation of the chromatin result in a template which is incapable of supporting RNA synthesis. In other cell systems(nucleated erythrocytes, lymphocytes) long-term inactivation of entire genomes is associated with a marked condensation of interphase chromatin. Inactivation of parts of a genome is normally associated with condensation of part of the interphase chromatin into heterochromatic clumps. Examples of this are the chromatin clumps of cells from the male mealy bug [l] and the sex chromatin of female mammalian cells. The authors thank Miss Nini A. Rosenfeld, Mrs Ulla Krondahl and Mrs Gertrud Blomgren for their skilful assistance. The development of the biophysical instruments used in this investigation was supported by grants from the Swedish Medical Science Research Council and the Wallenberg Foundation to Professor T. Caspersson. These studies were supported by funds from the Swedish Natural Science Research Council, Swedish Agricultural Research Council, the Swedish Cancer Society, the USPHS (HD-03577 and HD-14,733) and the Pennsylvania Fair Fund (ME-95).

REFERENCES 1. Berlowitz, L, Pallotta, D & Sibley, C H, Science 164 (1969) 1527. 2. Bishon. M W H & Walton. A. Marshall’s vhvsioloi of reproduction (ed’A S Parkes) vol. i, D. 2. 3rd edn. Lonamans &Green. London (1960). 3. Bolund, L, Ring&z, N R & Harris, H,‘J ceil sci 4 (1969) 71. 4. Bril-Peterson, E & Westerbrink, H G K, Biochim biophys acta 76 (1963) 152. Exptl Cell Res 62

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5. Caspersson, T 0 & Lomakka, G M, Instrumentation in biochemistry (ed T W Goodwin) vol. 26, p. 25. Academic Press, New York (1966). 6. Chamberlain, P J & Walker, P M B, J mol biol 11 (1965) 1. 7. Chargaff, E, The nucleic acids (ed E Chargaff & J N Davidson) vol. 1, p. 307. Academic Press, New York (1955). 8. Dallam, R D & Thomas, L E, Biochim biophys acta 11 (1953) 79. 9. Dariynkiewicz, Z, Gledhill, B L & Ringertz, N R, Exptl cell res 58 (1969) 435. 10. Gledhill, B L, The testis (ed A D Johnson, W R Gomes & N L VanDemark) Academic Press, New York (1970). In press. 11. Gledhill, B L, Dariynkiewicz Z & Ringertz, N R, J reprod fertil. In press. 12. Gledhill, B L, Gledhill, M P, Rigler, R & Ringertz, N R, Exptl cell res 41 (1966) 652. 13. Kernel& A M & Ringertz, N R, Exptl cell res. To be published.

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14. Kossel, A, Z physiol Chem 8 (1884) 511. 15. - The protamines and histones. Longmans & Green, London (1921). 16. Lindstriim. M. Zetterbere. A & Carlson.7 L., Exptl cell res 43 (1966) 537. 17. Mann. T. The biochemistrv of semen and of the male reproductive tract, p. 347. Methuen, London (1964). 18. Monesi, V, Exptl cell res 39 (1965) 197. 19. Rigler, R, Acta physiol Stand 67, suppl. 267 (1966). 20. Rigler, R, Killander, D, Bolund, L & Ringer& N R, Exptl cell res 55 (1969) 215. 21. Ringertz, N R & Bolund, -L, Exptl cell res 55 (1969) 205. 22. Ringertz, N R, Bolund, L & Dariynkiewicz, Z, Exptl cell res. To be published. 23. Co-urot, M, Hochereau, M T & Ortavant, R, The testis (ed A D Johnson, W R Gomes & N L VanDemark). Academic Press, New York (1970). In press.