Differential Activity and Synthesis of Lactate Dehydrogenase Isozymes ...

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,

REFERENCES

are expressed (reviewed

Hecht,

mouse

cDNA probes are being used to differential expression of LDH-A,

Akai

both the germ cell-specific isozymes during germ cell expression of the germ

period

1956)

cloned

179

ACKNOWLEDGMENTS

cell-specific LDH-C4 is initiated during meiosis, somatic-type LDH isozymes in meiotic and haptoid cells apparently do not undergo rapid turnover. The persistence of the somatic-type isozymes throughout sis (Oakberg,

the

cytochrome

examples

isoforms that spermiogenesis

current study examines and somatic-type LDH differentiation. Although

most

associates,

separated heterote-

LDH-C subunit are and Hawtrey, 1967;

kinase-2,

histone

cell-specific protein during meiosis and/or Willison

his

CELLS

1968).

phosphoglycerate

a-tubulin

are

initiation

with

IN SPERMATOGENIC

during

synthe-

cells

the mouse must be temporally or spatially from LDH-A and LDH-B synthesis, since trameric isozymes containing not formed in vivo (Goldberg

from We

or haploid if not all, of the

is consistent

in this

the

germ to

C ISOZYMES

cells 1977b).

in meiotic that most,

spermatogonia

This

conclusions

B AND

all other somatic (Bellv#{233}et a!.,

sis of the somatic type germ cells. Our results the

LDH-A,

Carr

JE, Olds-Clarke spermatozoa from

P,

Storey inbred

BT, and

1981. random

Oxidative metabolism bred mice. J Exp

of zool

216:285-92 DW, Usselman

MC, Acott TS, 1985. Effects of pH, lactate, and viscoelastic drag on sperm motility: a species comparison. Biol Reprod 33:588-95 Clermont Y, Trott M, 1969. Duration of the cycle of the seminiferous epithelium in the mouse and hamster determined by means of #{176}H-thymidine and radioautography. Fertil Steril 20:805-17 Cooper TG, 1984. The onset and maintenance of hyperactivated motility of spermatozoa from the mouse. Gamete Rca 9:55-74 Eddy EM, O’Brien DA, Rockett DL, Chapman LM, 1986. Appearance of flagellum cytoskeletal antigens during spermatogenesis. J Cell Biol 103 :82a Eventoff W, Rossmann MG, Taylor SS, Torff HJ, Meyer H, KeiI W, Kilts HH, 1977. Structural adaptations of lactate dehydrogenase isozytnes. Proc NatI Acad Sci USA 74:2677-81 Fenderson BA, Toshimori K, Muller CH. Lane TF, Eddy EM, 1988. Identification of a protein in the fibrous sheath of the sperm flagellum. Biol Reprod 38:345-57 Fukasawa KM, Li SSL, 1987. Complete nucleotide sequence of the mouse lactate dehydrogenase-A functional gene: comparison of the exon-intron organization of dehydrogenase genes. Genetics 116 :99-105 Galdieri M, Ziparo E, Palombi F, Russo MA, Stefanini M, 1981. Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions. J Androl 5:249-54 Gerton

GL, Millette CF, 1986. Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture. Biol Reprod 35:1025-35 Goldberg E, 1965. Lactate dehydrogenase in spermatozoa: subunit

180

LI ET AL.

interaction in Vitro. Arch Biochem Biophys 109:134-41 Goldberg E, 1972. Amino acid composition and properties of crystalline lactate dehydrogenase X from mouse testes. J Biol Chem 247:2044-48 Goldberg E, 1973. Molecular basis for multiple forms of LDH-X. J Exp Zool 186:273-78 Goldberg E, 1977. lsozymes in testes and spermatozoa. lsozymes Curt Top Biol Med Res 1:79-124 Goldberg E, Hawtrey C, 1967. The ontogeny of sperm specific lactate dehydrogenase in mice. J Exp Zool 164: 309-16 Goldberg E, Hawtrey C, 1968. The effect of experimental cryptorchism on the isozymes of lactate dehydrogenase in mouse testes. J Exp zool 167:411-18 Grootegoed JA, Jansen R, van der Molen HJ, 1984. The role of glucose, pyruvate and lactate in ATP production by spermatocytes and spermatids. Biochim Biophys Acta 767:248-56 Grootegoed JA, Jansen R, van der Molen HJ, 1986. Effect of glucose on ATP dephosphorylation in rat spermatids. J Reprod Fertil 77 :99-107 Hawtrey C, Goldberg E, 1968. Differential synthesis of LDH in mouse testes. Ann NY Acad Sci 151:611-15 Hecht NB, 1987. Detecting the effects of toxic agents on spermatogenesis using DNA probes. Environ Health Perspect 74:31-40 Hints M, Goldberg E, 1977. Immunohistochemical localization of LDH-X during spermatogenesis in mouse testes. Dcv Biol 57: 375-84 Holbrook JJ, Liljas A, Steindel SJ, Rossmann MG, 1975. Lactate dehydrogenase. In: PD Boyer (ed.), The Enzymes. New York: Academic Press, pp. 191-292 Jutte NHPM, Jansen R, Grootegoed JA, Rommerts FFG, Clausen OPF, van der Molen HJ, 1982. Regulation of survival of rat pachytene spermatocytes by lactate supply from Sertoli cells. J Reprod Fertil

round Growth Nakamura

NHPM, Jansen R, Grootegoed JA, Rommerts FFG, van der Molen HJ, 1983. FSH stimulation of the production of pyruvate and lactate by rat Sertoli cells may be involved in hormonal regulation of spermatogenesis. J Reprod Fertil 68:219-26 Lacmmli UK, 1970. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature (Lond) 227:680-85 Yaun Jl-l, Goldberg E, 1982. Lactate dehydrogenase isozymes from mouse. Methods Enzymol 89:351-58 Lessley BA, Garner DL, 1983. Isolation of motile spermatozoa by density gradient centrifugauon in Percoll. Gamete Res 7:49-61 Li SSL, 1988. Lactate dehydrogenase isoenzymes of mammals and the Lee

C-Y,

genes

coding for these enzymes. Riochem Soc Trans (in press) Fitch WM, Pan YCE, Sharief F5, 1983. Evolutionary relationships of vertebrate lactate dehydrogenase A4 (muscle), B4 (heart) and C4 (testis). J Biol Chem 258:7029-32 Markert CL, Shakelee JB, Whitt GS, 1975. Evolution of a gene. Science 189:102-14 Meistrich ML, Trostle PK, Frapart M, Erickson RP, 1977. Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes. Dcv Biol 60:428-41 Mita M, Hall P, 1982. Metabolism of round spermatids from rats: lactate as the preferred substrate. Biol Reprod 26:445-55 Nakamura M, Kamachi T, Okinga S, Arai K, 1986a. Metabolism of

Li SSL,

pyruvate

Differ 28:489-98 M, Okinaga S,

Arai

cannot K,

maintain

1984a.

the

Metabolism

ATP of

level.

Dcv

pachytene

primary spermatocytes from rat testes: pyruvate maintenance of adenosine triphophate level. Biol Reprod 30:1187-97 Nakamura M, Okinaga 5, Arai K, 1984b. Metabolism of round spermstids: evidence that lactate is preferred substrate. Am J Physiol 247:E234-42 Nakamura M, Okinaga 5, Arai K, 1986b. Studies of metabolism of round spermatids: glucose as unfavorable substrate. Biol Reprod 35 :927-35 Oakberg EF, 1956. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am J Anat 99:507-16 O’Brien DA, 1987. Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse. Biol Reprod 37:147-57 O’Brien DA, Fr#{248}ysa A, Rockett DL, 1986. Sertoli cell-conditioned medium increases viability and ATP levels of pachytene spermatocytes and round spermatids in vitro. J Cell Biol 103 :485a Robinson R, Fritz lB. 1981. Metabolism of glucose by Sertoli cells in culture. Biol Reprod 24:1032-41 Romrell U, BellvC AR, Fawcett DW, 1976. Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dcv Biol 49:119-31 Sakai I, Sharief FS, Li SSL, 1987. Molecular cloning and nucleotide sequence of the cDNA for sperm-specific lactate dehydrogenase-C from mouse. Biochem J 242:619-22 Storey BT, 1980. Strategy of oxidative metabolism in bull spermatozoa. J Exp Zool 212:61-67 Storey

BT, Kayne FJ, 1977. Direct intramitochondrial mitochondria. Biol Reprod

Storey

BT, Kayne FJ, 1978. Energy metabolism Interactions between lactate, pyruvate substrates for rabbit sperm mitochondria.

65:431-38 Jutte

spermatids:

Energy metabolism lactate oxidation 16:459-556

of

spermatozoa. by rabbit

VI. sperm

of spermatozoa. VII. and malate as oxidative Biol Reprod 18:527-36

SungWK, Komatsu M, Jagiello GM, 1986. The kinetics of the first wave of spermatogenesis in the newborn male mouse. Gamete Res 14:245-54 Tanaka S, Fujimoto H, 1986. A postmeiotically expressed clone encodes lactate dehydrogenase isozyme X. Biochem Biophys Res Commun 136:760-66 Wheat TE, Goldberg E, 1983. Sperm-specific lactate dehydrogenase C4: Antigenic structure and immunosuppression of fertility. Isozymes Curt Top Biol Med Res 7:113-30 Wieben ED, 1981. Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse. J Cell Biol 88:492-98 Willison K, Ashworth A, 1987. Mammalian spermatogenic gene expression. Trends Genet 3:351-55 Wistow GJ, Mulders JWM, dejong WM, 1987. The enzyme lactate dehydrogenase as a structural protein in avian and crocodilian lenses. Nature (Lond) 326:622-24 Wu KC, Chan K, Lee C-YG, Lau Y-F, 1987. Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-x gene. Biochem Biophys Res Commun 146:964-70