Histones of Rat Testis Chromatin During Early Postnatal Development ...

BIOLOGY

OF REPRODUCTION

17, 760-768

Histones

(1977)

of Rat Testis

Development NATHANIEL

Chromatin

and Their

C. MILLS2,

NGUYEN

T. VAN

Department Baylor

During

of Cell College

Houston,

Early

Interactions

with

and

Postnatal DNA’

ANTHONY

R. MEANS

Biology,

of Medicine, Texas

77030

ABSTRACT in the rat testis during early testicular development have been analyzed on acid-urea polyacrylamide gels and amounts of the individual histones quantified at 5 day intervals from 5 through 35 days of age. Three new histones, FIM, F2a2M and F2bM as first reported by Branson et al. (1975) are shown to originate from the primary spermatocyte and displace somatic histones Fl, F2a2 and F2b, respectively. Small bands of modified histones which are derivd from the mitotically active supporting (immature Sertoli cells) cells are present at 5 and 10 days of age, but disappear by 20 days of age, a time coincident with cessation of replication of the Sertoli cells. Testis preparations having no germinal epithelium (SCE-testis), have only somatic type histones, thus providing further support for the germ cell origin of the FM histones. Finally, hyperchromicity profiles of rat testis chromatins reveal that chromatin preparations containing the new meiotic histones are thermally more stable. These data suggest a tighter binding of the new histones to the DNA of meiotic cells. The

histones

present

INTRODUCTION

Histones, ar

DNA

the

and

repressors from (Hnilica,

been differ

in

basic

thought

to

tissues histones

in of

germinal et al.,

epithelium 1973; Branson

1975;

Shires

not

clearly In

the

are

quite

the same germinal

in plant microspores have revealed new

et

similar

1975).

type of the these new

However,

subsein the

seminiferous proteins

are

the

study,

histones

were

the

Sertoli

is

35

days

on

acid-urea

quantitation represented

the

testes

do

and proportional developing rat

not

denaturation

(SCE) of

The

polyacryl-

and changes were then

was

ani-

age. scans

of the area the different

cell-enriched

from of

spectrophotometric

al., 1975) the origin

thermal

established. present

appearance cells in

prepared

and

analyzed

and

the the

these

par-

epithelium derived

chromatin 5

were gels

dali et establish

of several species (Strokov et al., 1975; Grimes et al.,

al.,

testis

bands. The appearance particular histone bands

and

and histones

between

analyzed by peak which

which of the

Sheridan

rat

amide

species. These cells were first by

from mals histones

gene

phylogeny tissue has

histones histones

biochemically

(1967) studies

cell which

to nucle-

nonspecific

specific the

have

Stern quent

ticular from

be

tissues,

from

demonstrated

bound

specie throughout In contrast, germinal

markedly

somatic unusual

to

somatic

specie to 1972). shown

proteins

were

under the histone

occurring correlated

populations of testis. Further, rat

testis

(Tin-

used to more the new histones

contain studies

in to

germ were

cells.

clearly since Finally,

performed

to

explore possible changes in the derivative files of hyperchromicity vs temperature chromatins brought about by the presence the new histones on DNA.

proof of

isolated MATERIALS

AND

METHODS

Chemicals Urea, sucrose and Tris (ultra pure) were obtained from Schwarz/Mann Biochemicals. Acrylamide, N,N’bisacrylamide, N,N,N’,N’-tetramethylethylenediamine (TEMED), and Naphthol blue-black (Color Index

Accepted July 18, 1977. Received May 18, 1977. ‘Submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree (Nathaniel C. Mills), Vanderbilt University, Nashville, Tennessee. address: Department of Medicine, Endocrinology Division, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033.

#20470) were purchased from Eastman Organic. Both the acrylamide and bisacrylamide were recrystallized from acetone before use. Pyronin B (Color Index #741) was obtained from the National Aniline Division of Allied Chemicals. Ammonium persulfate and cacodylic acid were purchased from Fisher Scientific

760

HISTONES

OF

THE

DEVELOPING

Co. The cacodylic acid was recrystallized from ethanol before use. (Ethylenedinitrilo) terra-acetic acid (EDTA) was from Mallinckrodt. Silicone oil (DC-200) was from J. T. Baker Co. and helium gas (chromatographic quality) was from Big Three Industries. Highly polymerized salmon sperm DNA was obtained from Calbiochem. Animals Male rats were obtained from the Holtzman Co. were purchased at specified ages, body weights weight ranges. These specifications were envoked to assure a more consistent cell population in the rat testes at specific ages (Mills et al., 1977). Pregnant female rats purchased from Holtzman at 14 days of gestation were X-irradiated with 125 rads from a cobalt source on Day 20 of gestation, according to Tindall et al. (1975). Two days after parturition, the and and

young male rats were combined to form a litter size of 6 pups per lactating mother. The testis tubules of these animals are devoid of germ cells. These Sertoli cell enriched testes are abbreviated in this communication as SCE-testes.

Nuclear

Animals were killed and testes were immediately removed and placed in cold (0-4#{176}C) homogenization buffer (0.32 M sucrose, 50 mM Tris, pH 7.5, and 3 mM MgCl2. After removal of the runical albuginea, the tissue was minced, diluted with 20 volumes of homogenization buffer and homogenized using a Dounce homogenizer. The homogenate was filtered through three layers of organza cloth, centrifuged at 1000 g using the Sorvall HB-4 swinging bucket rotor and the nuclear pellet was resuspended in 10 volumes of 2.2 M sucrose containing 50 mM Tris, pH 7.5, and 3 mM MgCI2. The nuclei were pelleted from the 2.2 M sucrose by centrifugation at 15,000 g for 45 mm. The resulting nuclear pellet was used to prepare chromatin.

Chromatin

Preparation

Chromatin

was prepared by the procedure of (1971). Nuclei were suspended and lysed in 20 volumes of 0.08 M NaG, 0.02 M EDTA, pH 6.3, by several passes in a loose fitting Teflon-glass homogenizer. The chromatin was pelleted by centrifugation at 5000 g for 15 mm and the NaCl-EDTA wash was repeated followed by centrifugation. The pelleted chromatin was then washed in 20 volumes of 0.3 M NaCI and again pelleted by centrifugation at 5000 g for 15 mm. After a repeat of the 0.3 M NaCI wash and centrifugation the chromatin was resuspended, allowed to hydrate in 1.0 mM Tris, 0.1 mM EDTA, pH 7.5, and centrifuged at 5000 g for 15 mm. The chromatin pellet was resuspended in the above hydration buffer (1 mI/gm tissue) and stored in liquid nitrogen until used.

Spelsberg

Chromatin

Analysis

and nonhistone chromosomal protein conof chromatin were assayed in duplicate samples of 0.5 ml of chromatin (0.2-0.4 mg DNA). Samples were diluted to 1.0 ml with H2O and brought to 0.4 N acid Histone

tent

by

slowly

adding

4.4

N

H2S04.

The

histones

were

TESTIS

761

extracted for 10 mm at 0#{176}C followed by centrifugation at 1000 g for 10 mm to pellet the acid insoluble material. The supernatant containing the histones was removed and adjusted to an alkaline pH by addition of 0.1 ml of 5.0 M NaOH. The acid insoluble material was suspended in 1.0 ml of 0.1 N NaOH and aliquots removed for protein determination. Proteins were assayed as described by Lowry et al. (1951) using bovine serum albumin as a standard. The yield of chromatin per gram of tissue at 5 or 10 days of age was 4 times greater than that obtained from rat testes at 35 days of age. The decrease in chromatin yield is partially accounted for by the decrease of the DNA content of the testes during development which is approximately 8 mg DNA/gm tissue at 5 days and 2 mg DNA/gm tissue at 35 days of age (Mills et al., 1977). The protein to DNA ratio from the isolated chromatins was slightly less than 2:1 throughout testicular development. The total histone to DNA content ranged from 1.00 to 1.22 mg histone/mg DNA (average 1.13). The nonhistone chromosomal proteins averaged 0.62 mg protein/mg DNA and ranged from 0.5-0.7. Isolation

Isolation

RAT

of

Total

Histones

minimum of 500 g of DNA as chromatin was with 2 ml of 0.4 N H2 SO4 for 10 mm at 0#{176}C in accord with the procedure of Fambrough and Bonner (1966). The acid insoluble material was pelleted by centrifugation at 1500 g for 10 mm. The supernatant was dialyzed against 100 volumes of cold deionized H2 0 with two changes at 2-4 h intervals. The dialysate was placed in small vials, shell frozen, lyophilized and stored at -20#{176} C in sealed vials. To prepare the lyophilized histones for electrophoresis on acid-urea polyacrylamide gels, the histones were dissolved in a small volume of distilled water and assayed for protein concentration by the method of Lowry et al. (1951). Duplicate samples of 15-20 g of total histones were mixed 1:1 (v:v) with 4.0 M urea containing 1 percent 2-mercaptoethanol and allowed to stand at room temperature for 30 mm to assure reduction of F3 sulfhydryl linkages. Ten microliters of 0.1 percent Pyronin B were then added to each sample A

extracted

to provide

a front

marker.

Gel Electrophoresis Histones were separated in 6 mm (diameter) by 10 cm (length) 15 percent polyacrylamide gels with 0.1 percent N,N’-bisacrylamide as a cross-linker and containing 2.5 M urea and 0.9 N acetic acid as described by Panyim and Challdey (1969). After polymerization with ammonium persulfate and TEMED the gels were pre-electrophoresed at 2-5 mAmps/gel. The prepared histone samples were then applied and separated by electrophoresis at 1.5 mAmps/gel. Samples were run at room temperature until the dye band migrated off the bottom of the gel. The histone gels were immediately removed from the tubes and placed in 40 percent methanol, 10 percent acetic acid, 0.1 percent Napthol blue-black and 50 percent distilled water. The gels were stained 4-6 h and then destained in several changes of 40 percent methanol, 10 percent acetic acid and 50 percent water. After destaining, the histone gels were scanned at

MILLS

762

600 nm using a GCA/McPherson recording spectrophotometer equipped with a linear transport. Since each histone was reported to bind Napthol blue-black in proportion to the amount of histone present (Wright and Olins, 1975), the histone fractions were quantitated by Xeroxing the gel scans, cutting out each peak and weighing the paper on a semimicro balance. The amount of dye bound to each histone band was found to be linear for 5-20 g of total histones from rat testis 20 days of age. Nonlinearity of dye binding was observed when 40 g or more of the rat testis histone was applied to each gel. Of the different histones, F2a1 exhibited the greatest variation in dye binding.

DNA

Purification

Rat testis DNA was purified according to Marmur (1961), with the addition of a-amylase at the RNA5e step to remove polysaccharides. DNA content was measured by the procedure of Burton (1956).

Hyperchromicity Thermal performed

denaturation using

a

of rat

testis

modification

chromatins

of

the

was

procedure

12

ET AL described

by

Ansevin

and

Brown

(1971).

Approxi-

mately 100 g of DNA or DNA as chromatin was diluted to 1.25 ml using a solution of 1.0 mM Tris-HC1, pH 7.5, and 0.1 mM EDTA. To this sample an equal volume of freshly prepared thermal denaturation buffer (7.2 M urea, 0.01 M cacodylate, pH 7.0, 0.6 mM EDTA) was added and followed by vortexing to mix the chromatin and buffer thoroughly. The chromatin samples were then sheared at 5000 psi in a small (5 ml) French pressure cell which had been precooled (4#{176} C). The sheared chromatin was centrifuged at 5000 g for 15 miii at 4#{176}C to remove insoluble particulate matter. Ten to 15 percent of the chromatin was not solubilized as determined by A260nm measurements. The sheared chromatin was adjusted to an A260nm of 0.4-0.7/mi to achieve an optimum absorbance for thermal denaturation. The chromatin solution was degassed with helium for 5 miii to avoid bubble formation in the cuvettes upon heating. A small amount of helium in the sample does not interfere with denaturation since this gas has a negative coefficient of solubility with temperature. After placing the chromatin solutions in the cuvettes, the samples were overlayered with silicone oil and the cuvettes capped with teflon stoppers. Thermal denaturation was performed in a spectrophotometer which monitored 3 samples and a blank for absorbance changes at 260 nm. The temperature of the samples was increased at a linear rate of 0.8#{176}C/mm with a bath which circulates a (progressively) heated solution of ethylene glycol through the thermal spacers of the cuvette housing. The temperature change was monitored with a thermometer immersed in the blank cuvette and was recorded as each sample was read. Each chromatin sample was corrected for thermal expansion. The data, recorded on tape, were analyzed by a computer which calculated the hyperchromicity (A260nm T#{176}/A260nm initial) and the first derivative of the hyperchromicity vs temperature.

RESULTS

Comparison Cell

of Somatic

Histones

Fl

_____

gel Fl,

electrophoresis. F3, F2b, F2a2

F2a1 F2a1

FIG. 1. Analysis of histones from somatic and germinal tissues of rats on acid-urea polyacrylamide gels. Histones 20 Mg/gel were separated on 6 mm diameter by 10 cm long gels. The histone samples are: (1) rat liver histones and (2) 20 day old rat testis histones.

isolated

1) and testis and analyzed

Germ

are

The opment

as than

1, gel

1).

nomenclature

of Testicular somatic (Fig.

2)

liver

(Fig.

1,

of 20 day polyacrylamide

old

small

(Johns,

bands

the

unmodified

The

testis 1,

migrating F3

had

at

gel

2)

and

least

2

when

Development

histones 2)

are as

nomenclature of Johns rat liver histones F3 and

extra histone bands (Fig. compared to liver histones. Histones

rat

1, gel acid-urea

The rat liver histones and F2a1 (top to bottom)

observed slower

(Fig.

from

(Fig. by

by the modified

The

slightly

F2AI-.

were

gel rats

designated (1969). F2A2

and

Histones

are

of rat

testicular

designated

by

the

the

germ

1969)

and

devel“F” cell

HISTONES

5 10

15

20 25

3035

-

-

OF THE

DEVELOPING

RAT

763

TESTIS

-

F2A1.ip

FIG. histones of

2a. Polyacrylamide during development.

gel profiles of rat testes The numbers at the top

gels designate the age in days of the rats. histones are labeled on the left and histones on the right.

the

somatic meiotic

histones

are

designated

by

the

“F”

The the

nomen-

clature followed by “M” (meiosis) to indicate the somatic histone which is being replaced by each germ cell histone. Testes of 5 and 10 day old rats (Fig. 2a) which are similar of rat located Fl are

have identical to the somatic

histone histone

profiles profile

liver. Three small bands of proteins just above the major histone fraction observed and the 2 bands closest to Fl,

designated

as

Fl’

and

Fl”,

are bands

the gels as well F2a1

demonstrable as broadening on the spectrophotometric

(Fig. 2b). While as the shoulders disappear

protein at 3.6 profiles The

band

by

the of

20

which

days

the

and F3

Fl” and

is also

height sponding

and

band

F2b.

chronologically

evident

width diminution The

of

histone of the

appearance and

the

the

of amounts

histones

small

with

above

Fl

(peak

quantitation

testes

from

ent

the

in the

histone

at

days

15

increasing F3 with somatic

of

the

new of

peak a correhistone histones

the

differ-

age

and

their

become of

specified

age contain 2 new bands, F1M and F2a2M, which are clearly separated from the somatic histone bands (Fig. 2a and 2b) and are located just above Fl and F3 respectively. Histone F2bM

5

6

7CM

HISTONES

FIG. 2b. Spectrophotometric scans of the histone gels shown in Fig. 2a. Histone gels were stained with Napthol blue-black and scanned at 600 nm using a 0.2 mm slit width. The cm scale at the bottom shows the distance from the top of the gel.

age,

of

is located

from

of the scans of

bands Fl’ histones

cm in Fig. 2b) is present through 20 days. histones

4 TESTIS

RAT

electrophoretically

migrate as modified forms of Fl. At 5 and 10 days of age, the testis histones F3 and F2a1 have small, electrophoretically slower bands which larger

3

age

Quantitation

the

of

the

changes quanti7 and Fl at

later

different

from

histones

the at

scanned determined

during

development

duplicate chromatin on polyacrylamide and the in order

areas to

of the quantitate

prepagels, peaks the

with age (Fig. 3a and 3b). The of the histones, designated Fl, ‘at 5 and 10 days of age or Fl and ages,

the

Histones testis

were prepared from rations, separated stained, were

changes

apparent

intervals. of Testis

Histones

corresponding

more

does

not

change

substantially

total Fl’ F1M

MILLS

764

ET AL,

30

A

B

25

F3+F2BM

30. FI+FIM

w

20

z

25

0

I(I,

I

Lii

15

0-

-J

20

-

I-

I0

F2 B

C’)

I-

10

U-

‘5

0

F2AI

I-

0 I-

Fl’

5

10

#{149} #{149}FI

0

I

‘

I

I

5

10

15 DAYS

I

20

25

OF

AGE

I

30

I

35

40

FIG. 3a. Quantitation of the Fl histones during rat testicular development. Fl (0) and Fl” () are modified histones and F1M (.) designates the meiotic histone. The total Fl histone () is the sum of all Fl components.

during

testicular

percent histones The

12

Fl’

and 3 percent from testis, sum

of

between Fl’ and

Fl,

and

Fl”

respectively at 5 an, 10

Fl

and

Fl

3a).

The 5

represent

of the total days of age.

declines

At bands

from 15 10 days

percent of age

total

histones

and

at

amounts

of the

respectively from the histone

of testis

F2b

continues

peak

at F3

plus

32 30

days. F2bM

age,

the

7.5 percent to increase which time

more

than

12

10 days represent

an initial

of age the histone 25 and 21 percent extracted amount

decrease

percent

of the

The

observed diminishes

25

30

35

40

OF AGE histones other the presence of cells while the of the somatic

observed Histone

of the total histone to slightly less than

days of age. The histone at 15 days and migrates

(labeled F2a2M) increases cent of the total histones Thus, the appearance and F2a2M

for F1M as Fl F2a2 decreases

corresponds of

closely

histone

F2a2.

proteins at 10 percent

band which just above F3

to represent by 30 days increase of with

the

Histone

5 perof age. histone

decreased F2al

repre-

sents does

about 15 percent of the total histones and not appear to change substantially during

early

testicular

development.

of

at 15 days

days F2b represents 16 histones. The peak of F3 initial increase at 15 days

as F2b

by 30 appears

amount

the total histones (Fig. 3b). The

shows

represents

protein

of

histones.

of age and by 30 percent of the total plus F2bM shows an and

days

to slightly

total

5 and and F3

15

and represents F1M continues days of age at

20

FIG. 3b. Quantitation of rat testis than Fl. The closed symbols designate histones contributed by the meiotic open symbols represent quantitation type histones.

of the

fraction both F2b

15 DAYS

the increase (Fig. 3a).

Concomitantly,

percent

10

rapid as decreases

histone F1M appears of the total histones. in quantity until 25 this

5

rapidly

to decrease in quantity to 8 percent of the total histones by 25 to 30 days of testicular development.

O

and 15 days of age while the bands disappear. At 15 days of age Fl is

10 Fl”

percent

(Fig.

development

labeled

histones

F2A2M

5

total

is not

from

To clarify to conclusively the

nongerminal

of

from

SCE

as

electrophoresis.

histone

increase

Hi stones

Nongerminal

Cells

of

Testes

the origin of the new histones demonstrate the contribution cells, rat

histones

testes The

and

major

were analyzed

cellular

and of

prepared by component

gel

HISTONES

OF

THE

DEVELOPING

RAT

TESTIS

765

A

$

44,

I-)

5 7CM 4a. Profile of h(2) istones from normal and X-irradiated SCE testes. The gels are (1)histones from normal 20dFIG. ay old rat testes, and histones from 22 d ay old SCE testes. FIG. 4b. Spectrophotometric scan ofSCE testes histones. The cm scale shows the distance migrated from the origin. 3

5

4

U DAY

ITOPL3

1*

of

the

SCE

testis

is the

much smaller contributions cell and peritubular cells histones of

present

age

than

4a,

gel

1)

which histones

rat

(Fig.

4b)

corresponds

migrates (Fig.

at 2b).

3.6 cm However,

protein preparation

is substantially over that

cells

although

The

from the Leydig present. The new testes

are

histones (Fig. 4a, band which migrates Fl

days

are

in normal

(Fig.

SCE-testis protein

Sertoli

at 20 days

absent gel

from

2). The small slightly slower to

in

the

the

band

normal testes the amount of

enhanced of the normal

in the testes

SCE at 20

of age.

Thermal

Stability

During

Development

Histones volved such

are

thought

with the as folding

physical and

chromatin histones epithelium. studies yield and

of Testicular

preparaiions which are Therefore,

to

Chromatins

be

from the unique to thermal

of

extent

of histone

which histones

have were

binding

in-

the DNA Isolated

rat testes have the germinal denaturation

of chromatin were conducted information with respect to the

tin preparations containing only chromatins from

to DNA.

which nature Chroma-

from 5 and 10 day rat testis somatic type histones and 20, 25 and 30 day old rats

significant compared

amounts (Fig.

5, 6).

of

tained.

DNA

the

new

was

the temperatures of 54#{176}C(not Figure rat

found

to

intervals

temperature and 100

DNA denatured hyperchromicity

as a curves

testicular

chromatins

at least

3 inflection

are

C).

of ob-

between

from

were

developing

thermally

denatured

range While

of 50#{176}C(bepurified rat

monophasic obtained

curve, from the

multiphasic

points.

indicative

structure

amount

and

The

of (i.e.

and total

the rat had

hyper-

nature

total

differences

in

“packing”)

of the

5a, Sb). Since purified, DNA has a hyperchromicity the

first

(measured at 100#{176}C) for various samples range from 1.3 to 1.4. These

tertiary

1.40,

the

denature

isolated

at 5 day

a broad 50 C

values

and

vs temperature testis DNA were

of 48#{176}Cand 62#{176}Cwith a Tm shown). In contrast, as shown in

chromatins

5,

testes

over tween

profile

of hyperchromicity purified rat

chromicity chromatin

intimately

structure condensation.

hyperchromicity

derivative extensively

proteins highly of

hyperchromicity

of the 1.39 in comparisons

first

for

5 chromatin

the

(Fig.

polymerized approximately

to of peak

plots

the

present

tins were normalized increase the validity derivative

the

and/or

rat

chromaorder

to in the

prepa-

rations. These data are presented in Fig. 6a and 6b. Plots for rat DNA are also included to allow direct comparison. The hyperchromicity profiles a

observed

as

peaks

in

the

inflections of chromatins first

derivative

of

the are plots

MILLS

766

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In this study, we show that the histone profiles from rat testes at 5 and 10 days of age are similar in both specific bands present and

4

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IS

005011070110

.0

TENPESAD0

00

50

40

t

60

the

70

60

TEMnSAT_

00

50

.C

are

tive

much

plot

more

of

easily

day

5

analyzed.

testis

The

chromatin

derivashows

a

of

histone

Fl’ and represents

histone

FIG. 5a and b. Hyperchromicity profiles of rat testicular chromatins during early development. Chromatins were denatured by continuous heating in low-ionic strength urea-cacodylate buffer, pH 7.0. The data were recorded and then processed by computer. The hyperchromicity plots show every point, however, only every fifth point is designated by a symbol.

and

amount

bands, histone

at both

small

5 and

traces

remain

small

bands

recognized

F2al

histones

are

also

in each

Fl”, 36 at

of exist

small

above the total

10

days

of

age

15

days

of age.

and

at

5 and

10

Fl Fl only

Further, of

F3

spectrophotometric

and scans

days

any of the bands of Fl histones are derived from the germinal unlikely since the interstitial cells of the testis constitutes percent tones

Two

as shoulders

in the

present

band.

immediately percent of

of age.

That

at 5 days of age epithelium seems and supporting more than 96

the total cell population and in a stochiometric relationship

histo

sharp decrease in the area of the peak between 70 and 85#{176}Cand increase in the area of the peak at 60#{176}Cwhen compared to 10 and 15 day chromatins (Fig. 6a). The derivative plots of

DNA. Even at 12 days of age the germinal epithelium is less than 22 percent of the total cell population of the testes and the resting primary spermatocytes are approximately 9

hyperchromicity

percent

are

at

almost

20,

identical

observed observed

at at

25

with

66#{176}C(Fig. 20-30 days

and only

30

days

slight

of

age

variations

old

rats

(Fig.

6b),

is stabilized

it is apparent by

A small

6b). The three peaks of age occur at 65#{176}C, shoulders histone the derivative plots of

75 C and 90 C. When the chromatins of 5 and 10 day old animals (Fig. 6a) are compared to the derivative plots of chromatins isolated from testes of 20-3 0 day peak

of

1957). these

5#{176}(i.e.,

that Tm

the

shifted

first from

011*

‘S

5007

o.oftTCft,0040

V

a

05

20

and

acetylation.

cause of

a slightly the specified

would mobility

and

these

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70

7

are

of

age.

histone

age.

I

0

days

very

active the

would

The

observation

been

shown

in preparation

for

et al., 1974; Borun et al., 1974; Tobey et al.,

cells

Thus,

bands

have

cycle

Clermont

that

modifica-

be

of

that

and

Percy

the

seminiferous

in cell

(1957)

division

presence

of

anticipated,

at 10 modified

since

dividing of the histones

and testis Fl’

the

are the at this and

Fl”

are present in the testis at 5 and 10 days of age agrees with the data of Grimes et al. (1975) but is in contrast to the reports of Shires et al. (1975). However, we propose that Fl’ and Fl”

#{149} Ii

60

cell

of

slower electrophohistones on gels

histones the

In addition, shown

These

supporting cells are actively major cellular component

‘II

40

modified

to occur

Percy, is that trailing

F2a1) are the result such as phosphorylation,

tions retic

epithelium

.-.tt

‘S

OO

00

and

and

explanation Fl” and the

methylation

1974).

00

(Clermont

plausible (Fl’,

of F3 modifications

have

#{163}

total

mitotic division (Allfrey al., 1974; Chalkley et

60#{176}C to 6 5#{176}C).

s-_SY. .._RTCSS

the more bands

are

#{149}016i TE05AT

#{149}C

FIG. 6a and b. Derivative plots of the hyperchromicity profiles of rat testicular chromatins during early development. The first derivative was calculated by computer from the hyperchromicity curves shown in Figure Sa and b. Every fifth observation is designated by a symbol. The first derivative plot of rat testis DNA is superimposed on each plot for reference.

derived

Grimes which We new

from

et are

somatic

cells

(in

contrast

to

al., 1975) and represent histones modified during the cell cycle.

show histones

evidence in are present cells

this work that when the rat

as has

been

reported

three testes

contain

meiotic

Branson Indeed,

et al. (1975) and Grimes et al. (1975). the new histone bands designated F1M,

by

1-IISTONES

F2a2M

and

F2bM

of age

in the

rat

primary numbers

first testes.

spermatocytes at 15 days

even

greater

appear

at about

Zygotene

and

are present of age and

proportion

population

at

20

of

and

25

days

of age

contribute

to

during

testicular

epithelium and

less

Percy,

1957).

that the new nal epithelium germ cells, though, spermatids

these

It

as

to

proliferate

is,

therefore,

histones originate since SCE testes, have only somatic

an

(Clermont

cell

cease thus

germinal concluded

difficult tion to

to assign the these specific

chromicity

profiles

SCE

in

effectively histones Although

with

the

be derived represents

spermatocyte F2bM are

since histones F1M, F2a2M and present only in the germinal epitheli-

urn are

and only when present. Further,

these number

histones correlate with of primary spermatocytes

the total cell centage of the ric relationship

site of primary

the primary spermatocytes increases in the amount

population. new histones to DNA

is in close attributable DNA content)

above to

from cell

Indeed, if the perhave a stoichiometas do the somatic

purified the

rat DNA

DNA, is

variations of the testes chromatins rats of

may the

be

thus,

due

histones,

derivative from to

the

observed

similar

to

DNA

histone-DNA thermal somatic (1971)

of

the

meiotic

complex

which

denaturation histone-DNA. have related

than each

most

histones.

plots observed 5, 10 and 15

Szopa (1974). The temperature hyperchromicity with the histones suggest that these with

that

with

of The

in the day old

reported

shift presence histones cell is more the

by

give stable

complex

histone polymerized

meiotic The the

at small active

the

germinal 4 percent

A small Fl

band

age) and histones.

this The

band extent

ment by matocyte

meiotic histones and the function

study. Hyperchromicity chromatins containing meiotic cells have

which

(5 and

is pronounced of somatic

somes division ance

or

derived or Sertoli when the a

large

10 days

of

in SCE-testes histone replace-

in the primary sperof these new his-

studies the new thermally

of spermain sperfor further

revealed histones more

complex. These new histones for the folding and condensation

for some other process which

which cell

migrates

represent

population

a

testis

is probably

cells

cell

F1M

cannot

the presence is consistent of somatic

immature supporting cell its presence is observed supporting

of

to

bands

epithelium of the

histones

by of

germinal of histones

similar these

this age. However, bands at this age mitotic population the

cells studies

derivation

from bands

of age

similar present suggest somatic

that from stable may of

spermatobe in the chromo-

part of the reductionresults in the appear-

of spermatids.

a to of

Ansevin and Brown peak of the derivative

plot to the interaction of a particular histones using reconstituted highly

that

the DNA which occurs in primary cytes. An additional role might formation of the tetrad of meiotic

(5#{176}C)in of new interact to

from the less than

of

be small

of the

DNA-histone be required

modifications

results

com-

tones during meiosis and later stages togenesis, such as histone replacement matids by protamines, remain topics

reveal very at 54#{176}Cas

suggesting

complexed

the since

portion

1957). profiles denatu-ring

are

interactions. been shown

mobilities 5 days

cells. all

immature

agreement to the amount of to primary spermatocytes at this age by cell count

(Clermont and Percy, The hyperchromicity little DNA of chromatin

at

supporting

histones, it can be calculated that at 30 days of age, 50 to 60 percent of testicular DNA is derived from the primary spermatocytes. This value DNA (4N

present

population of these with an

of

the increasing in relation

stabilizahyper-

chromatins

show to

electrophoretic

observations origin of

the the

DNA because

replaced in for meiosis.

testes

are

with being

from

these new epithelium.

since these cells are not present until meiosis is observed at about 25 days of age. Thus, these are consistent new histones

increased histones

histone-DNA study it has

type histones are histones specific

from the germiwhich have no histones. Albe present in spermatids

767

the rat testis contains somatic histones to rat liver as well as new histones not in somatic tissues. These experiments that three and possibly more of the

(Clermont

new histones may they cannot originate

TESTIS

posites of all the In the present

population the

RAT

DNA and purified histone fractions. Although the observed temperature shift is in an area indicative of Fl and F2b-DNA interactions, it is

cell

cells age and

the

development

continues

15 days pachytene

testicular

supporting days of

and

DEVELOPING

in significant represent

the

and Percy, 1957). The mitotic division at 16 less

OF THE

or

ACKNOWLEDGMENTS We wish to thank Ms. Nancy Hammond for assistance in preparing this manuscript. This work was supported by research grant HD-07503 from the NIH and the Baylor Center for Population Research and Studies in Reproductive Biology (HD-07495). A. R.

768

MILLS

Means ment

is the Award

recipient from

of

the

a Research

Career

Develop-

NIH.

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