Park, North. Carolina. 27709. ABSTRACT. Spermatogenic cells isolated from adult and ...... D. Shelby for reading the manuscript and. Ms. N. Gore for typing it.
BIOLOGY
OF
REPRODUCTION
40,
173-180
(1989)
Differential Activity and Synthesis of Lactate Dehydrogenase Isozymes A (Muscle), B (Heart), and C (Testis) in Mouse Spermatogenic Cells STEVENS
S.-L.
LI,”2
DEBORAH DOUGLAS
A. O’BRIEN,3 L. ROCKETT,3
Laboratory of Genetics2 Laboratory of Reproductive National
Institute
and and
Gamete Biology Developmental
of Environmental
National Research
ESTHER W. HOU,2 and E. M. EDDY3
Institutes
Triangle
Park,
JUN
VERSOLA,2
Section, Toxicology3
Health
Sciences
of Health North
Carolina
27709
ABSTRACT Spermatogenic examine sis.
cells isolated from
enzyme
The
activities
synthesis
and
and activity
leptotene and leptotene/zygotene ously. The LDH-C4 isozyme
adult
synthesis of
LDH-C4,
the the
spermatocytes prominent
was
prepubertal
and
of
lactate germ
mice
by
unit
dehydrogenase
cell-specific
prior to in pachytene
gravity
(LDH)
isozyme,
was
the mid-pachytene spermatocytes,
sedimentation isozymes
were
during
detected
earliest
in
stage of meiosis round spermatids,
shown
muscle-type
to exhibit LDH-A
and
comparable
amounts
heart-type
LDH-B
of five
tetrameric
five tetrameric LDH-1 (B4),
isozymes
formed
isolated
pre-
by
LDH
isozyThis is cells were
combination
of
subunits.
INTRODUCTION
In mammals, (LDH) isozymes,
LDH
to
reported previand condensing
spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. in addition, somatic-type mes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli further
used
spermatogene-
interval, lactate dehydrogenase -2 (A1 B3), -3 (A2 B2),
and
LDH-C4
activity
is not
detected
until
-4 (A3 B,), and -5 (A4), are found in various proportions among different somatic tissues, whereas the homotetrameric LDH-C4 isozyme is present only in mature testis and spermatozoa (Markert et at., 1975).
the presence of pachytene spermatocytes (14-16 days postpartum) (Goldberg and Hawtrey, 1967; Hawtrey and Goldberg, 1968; Wieben, 1981). The continuous production of LDH-B subunits in the cryptorchid mouse testis (Goldberg and Hawtrey, 1968) and the absence of C-B heterotetramers in
Although conversion (muscle),
all
species with multiple C-containing berg, 1973) led these investigators LDH-B subunits are synthesized
isozymes matic, and
possess distinct immunological
the LDH isozymes catalyze of lactate and pyruvate, the LDH-B4 (heart), and LDH-C4 physicochemical, properties (Holbrook
1975; Eventoff et at., 1977; and Goldberg, 1983; Li, 1988). During testicular development, -C genes examining tionated LDH-A age,
Li et
at.,
the
1983;
LDH-A,
enzyet at.,
within the spermatogenic
subunits
increase
during
In contrast, cells and has
-B, and
cells throughout 1977). Synthesis
spermiogenesis of the LDH-C
at.,
functional
1977)
1981) also spermatocytes
this
and have
in mouse stage
isozymes (Goldto conclude that only in somatic cells
LDH-C4 is confined to been detected immuno-
histochemicatly the mid-pachytene
are
LDH-B
testis.
Wheat
expressed differentially. Previous studies the activity of LDH isozymes in unfracmouse testes found that the proportion of subunits decreases between 2 and 10 days of
while
Accepted Received Reprint
the interLDH-A4 (testis)
spermatocytes beginning at of meiosis and in haploid
been detected and spermatids.
(Hintz subunit
LDH-C
and Goldberg, (Meistrich
mRNA
in isolated Prepuberta!
et
(Wieben, pachytene germ cell
populations containing a mixture of spermatogonia, pre-pachytene spermatocytes, and nongerminal cells showed no evidence of LDH-C synthesis (Meistrich et at., 1977). The present study compares the relative activities of the LDH isozymes in germ cell populations pun-
August 11, 1988. May 31, 1988. requests.
173
174 fied
LI ET AL.
from
prepubertal
1977a,b).
A
system
and
sensitive
currently
adult
mice
agarose
used
for
(Betlv#{233}et al.,
gel
electrophoresis
analyzing
human
also
(O’Brien, subunits
cultured 1987) throughout
investigation LDH
-
in the to
monitor meiosis
provides
isozymes
of
evidence
in germ demonstrates
[35S] methionine
synthesis of the and spermiogenesis.
consisting
units are present genesis. It also and synthesis tocytes prior
presence
that
cells
of
throughout that LDH-C4
appear to be initiated to the mid-pachytene
LDH This B sub-
spermatoactivity
twice
(1983). Sertoli
Animals CD-i Charles breeding
and
AND
washed
tation
male mice (>8 wk old) were obtained from River Laboratories (Raleigh, NC). The NIEHS colony, established with mice from the same
source, provided all prepubertal animals. Cell suspensions from the seminiferous were prepared from adult and prepubertal
epithelium mice
by
dissociation (Romrell et at., 1977a,b), using reduced concen(0.5 mg/ml) and trypsin (0.25 with trypsin were terminated by
adding 0.25 mg/mt I-S, Sigma Chemical
soybean trypsin Co., St. Louis,
inhibitor (Type MO). Germ cells
at defined stages of spermatogenesis were purified by unit gravity sedimentation (Bellv#{233}et a!., 1977a,b). A and 8-day-old
type B spermatogonia animals. Preleptotene
were obtained spermatocytes,
a population containing both leptotene and zygotene spermatocytes (leptotene/zygotene), and prepubertal pachytene spermatocytes were isolated from 17-dayold mice weighing 7-10 g. Pachytene spermatocytes, round spermatids, and condensing spermatids were isolated from populations, morphology,
adult animals. determined on were assessed
(Bellv#{233}et a!., 1977a,b). 2-3 times in enriched
Purities of the germ cell the basis of cell size and as described previously
Alt germ cells Krebs-Ringer
were washed bicarbonate
medium (Bellv#{233}et at., 1977a) and were stored at -70#{176} C prior to electrophoresis. Mouse spermatozoa from the cauda epididymidis and/or vas deferens were collected, filtered through
NY),
and
phosphate-buffered
isolated
with
into
from
were were
PBS,
/
17-day-old
mice
previously (O’Brien et at., 48 h, remaining germ cells
hypotonic cells cells
of
and
presence >1000 Products, cytes
sequential enzymatic 1976; Beltv#{233}et at., trations of collagenase mg/ml). Incubations
Type from
by
Sertoti These
To examine spermatogenic
Cell Preparation
Elmsford,
Ca2/Mg2-free
were
Incorporation
METHODS
(Tetko,
as described culturing for
removed
1981) and 3-5 days.
(P5SJmet) MATERIALS
in
cells
and cultured 1986). After
dishes,
in early spermastage of meiosis.
mesh
saline (PBS, pH 7.4) at 4#{176}C.In some experiments, spermatozoa were further purified by density gradient centrifugation in Percott (Pharmacia, Piscataway, NJ) according to the procedure of Lessley and Garner
were
somatic-type
predominantly
80-pm
washed LDH
isozyme patterns was successfully adapted in these experiments to separate all six mouse LDH isozymes. Spermatocytes (preleptotene, leptotene/zygotene, and pachytene populations) and round spermatids were
Nitex
lysis cultured scraped
and
(Galdieri
et
at.,
for an additional from the culture
stored
at -70#{176} C.
SJMethionine
LDH
Isozymes
the synthesis cells purified by
Sertoli
cells
were
of unit
LDH gravity
isozymes, sedimen-
at
32#{176}Cin the
cultured
of 100 pCi/ml [35 SI methionine with sp. act. Ci/mmot (NEG-009A, DuPont NEN Research Boston, MA). Adult pachytene spermatoand
round
spermatids
were
cultured
for
16 h in
serum-supplemented Eagle’s minimal essential medium (MEM) according to the procedure of Gerton and Millette (1986). Sertoli cells and earlier spermatocyte populations isolated from 17-day-old animals were cultured without h, respectively. labeled reduced
serum In all
methionine to 10 pM
(O’Brien, 1987) for culture experiments,
concentration (O’Brien, 1987).
in At
18 and 6 the un-
MEM was the end of
each incubation period, cells were harvested and washed three times at 4#{176}Cwith enriched KrebsRinger bicarbonate medium (Betlv#{233}et at., 1977a). Aliquots of each sample were counted in Aquasol (Dupont NEN Research Products) to determine relative incorporation of [‘ SI met. LDH isozymes labeled with [35S]met were partially purified by affinity chromatography using 5’-adenosine monophosphate
(AMP)
Sepharose
4B (Lee
et at.,
1982).
Gel Electrophoresis and
Autoradiography
LDH fraction denaturing
isozymes present in the soluble protein were separated on agarose gels in nonbuffer (pH 8.2) and activities were visu-
alized by nitro blue tetrazolium reduction (Paragon electrophoresis system and drogenase
isozyme
kit,
Beckman
to formazan lactate dehy-
Instruments,
MOUSE
Fullerton,
CA)
according
recommendations. to detect enzyme
Lactate activity
Relative
of
activities
mined with Instrument,
an UltroScan Gaithersburg,
To compare files, purified were separated as
the
to
LDH-A,
the
B AND
manufacturer’s
was used as the substrate unless otherwise noted. LDH laser MD).
isozymes
were
densitometer
enzyme activity with LDH isozymes labeled on agarose gels, activity
described
above,
and
C ISOZYMES
the
dried
were
then
I-I
LDH-
1
U)
w
LU
90%. The
of
agarose
C4
bodies 30% ‘
spermatids at steps 9-16 of spermiogenesis (Romrell et al., 1976). LDH isozymes of Sertoli cells and different popu-
4:..;O
65%
an
earlier
initiated. pachytene
be
1977),
these early two
stage
in
could of
which
spermatocytes
In
between previous
pachytene
the mid-pachytene chemistry (Hintz toring biosynthesis 1977). We activity and but also have populations
LDH-C4
was
first
detected
at
stage of meiosis by immunohistoand Goldberg, 1977) and by moniwith [3 HI valine (Meistrich et a!.,
have observed synthesis in detected this of
and to be
strains. studies,
preleptotene
high pachytene isozyme and
levels
of LDH-C4 spermatocytes, in highly enriched
leptotene/zygotene
the precise
synthesis
is
of this isozyme contamination levels cells.
contain
spermatocytes.
in by
of LDH-C4 Leptotene/
small
Furthermore,
meiosis. Previous investigations the testis concluded present only in somatic
invariant
do
numbers
of early pachytene spermatocytes because of overlapping size distributions of these two populations. However, preleptotene spermatocyte populations generally do not contain the substantially larger
in testis. Efficient detection between mouse strains probably results, since both seminiferous 1969) appear
that
interpreting of the
for low meiotic may
types
LDH-C4
high level minor
account earlier
in round
primary spermaand nongerminal
must be exercised in this study for identification
tene spermatocytes rather than variation accounts for these
Trott, 1981)
popula-
preparations
cell
activity was detected during prepubertal prior to the appearance of pachytene
(Clermont and activity (Wieben,
cell
detected
from the testes of 12-day-old animals, which do not contain pachytene spermatocytes (Bellv#{233} et at., 1977a; Sung et at., 1986). Activity increased markedly between 13 and 16 days of age, coincident with the appearance and increasing proportion of pachy-
duration mRNA
sensitivity
and spermatozoa fixed mouse testes
study,
Because of the spermatocytes,
zygotene
(Goldberg and Hawtrey, 1967; Hawtrey and Goldberg, 1968). In this study a sensitive, high-resolution agarose gel system with low background was used. LDH-C4 activity was detected in samples prepared
cycle LDH-C
assay
procedures (Eddy et although synthesis of in prepubertal germ cell
of 26% spermatogonia
cells (Meistrich et at., not contain LDH-C4.
this cell type in populations
appears after birth
Both
of spermatogenic
in condensing sections of
spermatogenic both
that 1981)
the
>80%).
tions used in this study may have contributed to the detection of LDH-C4 prior to the pachytene stage. Immunohistochemical techniques are not always adequate for detecting antigens that are labile or present at low levels. For example, an antigen recog-
Caution results of
of sperm in these
LDH-C4 were detected earlier development than in previous of LDH-C4 enzyme activity suggested (Wieben,
and
(purities
LDH-C4 development spermatocytes
(Bellv#{233}et al., 1977a, b). Results with samples and with isolated germ cells LDH-C4 appears prior to the mid-pachytene
1968;
Goldberg,
whole suggest
testis that stage of
examining LDH isozymes in that LDH-B subunits were cells (Goldberg and Hawtrey,
1973).
However,
in
this
study,
enzyme activity of the somatic type LDH isozymes was detected in all germ cell populations examined, except spermatozoa isolated from the excurrent ducts. LDH-i and LDH-2, which contain predominantly LDH-B subunits, were more prominent than isozymes rich in LDH-A subunits, particularly during meiosis and spermiogenesis. These enzyme activities cannot comparable
be
attributed levels
to of
Sertoli
LDH-1
cells, (B4)
and
which LDH-5
contain (A4).
MOUSE
Furthermore,
the
study eliminate the germ cell
virtually populations
have
not
dissociation
obtained
LDH-A
conclusive
and
synthesized
in
of meiosis.
procedures
used
evidence
isozymes suggest
LDH-B
subunits prior
hypothesis
that
the
for
in
synthesis
of
LDH-C
Hawtrey
and
LDH-C4, c,
Goldberg,
the
and
previous
subunits
and
Hit
are
in
Ashworth,
of the
1987;
34.5-day
of germ
1987).
These
suggest
and
results Ashworth,
has that
1987)
the complete However, the
finding identical tempting
that the duck and to LDH-B isozyme to speculate that
LDH-A
protein
may
in
The
tion LDH
of temporal genes.
In
previously.
isotype
switching
not
necessarily
tene stage genes also published).
the
We thank manuscript
stages of
gene
result
in
isoforms. the various stages recent
crocodile c-crystalline are (Wistow et al., 1987), it is the LDH-B as well as the some
activity
structural during
role
in
spermatogene-
for the mouse LDH-A, LDH-B, have been cloned (Akai et at., Fujumoto, 1986; Sakai et at., Li, 1988) and the complete LDH-A gene has been deter-
tissue-specific
collaboration
with
expression Dr.
M.
Simon
investigate -B, and
of spermatogenesis.
Northern
blot
analysis
studies confirm is detected
-B,
that prior
and
-C
further -C genes
The
prelim-
and
in situ
the expression to the pachy-
of meiosis and that the LDH-A and are expressed in germ cells (5, S.-L.
LDH-B Li, un-
Drs. Frank M. Johnson and Ms. N. Gore for
and typing
Michael it.
D. Shelby
for
reading
K,
of the and
Yagi
K, Tiano
Li SSL,
1985.
HF, Pan Isolation
YCE, Shimizu M, and characterization
Fong K, Jungmann of a cDNA and
a
pseudogene for mouse lactate dehydrogenase-A isozyme. Int J Biochem 17:645-48 Allen JM, 1961. Multiple forms of lactic dehydrogenase in tissues of the mouse: their specificity, cellular localization, and response to altered physiological conditions. Ann NY Acad Sd 94:937-5 1 Alvarez JG, Storey BT, 1984. Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa. Biol Reprod
30:323-31 Bellv#{233}AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar M, 1977a. Spermatogenic cells of the prepuberal mouse.
YM, Dym Isolation
morphological characterization. J Cell Biol 74:68-85 Relive AR, Millette CF, Bhatnagar YM, O’Brien DA, 1977b. Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem 25:480-94 Boell EJ, 1985. Oxygen consumption of mouse sperm and its relationship to capacitation. J Exp Zool 234:105-16 and
Carey
and Li, 1987). Such information on be useful in examining the regulaand
results
cytohybridization of the LDH-C
(Willison
later spermatogenic In light of the
play
1987; Wu et al., 1987; structure of the mouse wilt
specific
inary
RA,
spermatogene-
reported
of somatic significance of
LDH isozymes during the remains to be determined.
mined (Fukasawa gene structure
been
does
replacement metabolic
addition to enzymatic sis. Recently, cDNAs and LDH-C subunits 1985; Tanaka and
of mouse
not
LDH-A,
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ACKNOWLEDGMENTS
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in this
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