A study of rat epididymal sperm adenosine 3',5'-monophosphate-dependent protein kinases: maturation differences and cellular location

BIOLOGY OF REPRODUCTION 32, 155-171 (1985)

155

A Study of Rat Epididymal Sperm Adenosine 3’,5’-Monophosphate-DependentProtein Kinases: Maturation Differences and Cellular Location

R. W. ATHERTON,”2 S. KHAT00N,�P. K. SCHOFF4 and B. E. HALEY3

Department of Zoology and Physiology2

and Department of Biochemistry3

University of Wyoming

Laramie, Wyoming

and

Enzyme Institute4

University of Wisconsin

Madison, Wisconsin

ABSTRACT

The photoaffinity analog [32 P1 8-N3cAMP (8-azido adenosine 3,5’-monophosphate was used

to analyze the membrane sidedness of rat sperm cAMP binding proteins during epididymal matura-tion. Evidence is presented here -which supports the hypothesis that 3 5-45% of the regulatory

subunits of the Type I and Type II cAMP-dependent protein kinases are readily available to exter-

nally added cyclic nucleotide. It was observed by sodium dodecyl sulfate gel electrophoresis

(SDS-PAGE) and autoradiography that only two rat sperm proteins (Mr=49K and 55K) were photo-labeled which comigrated on gels with partially purified Type I and Type II regulatory subunits,

respectively. Both of these photolabeled epididymal sperm proteins were saturated at physiological

titers of [“P18-N3 cAMP and photoincorporation of [32 P1 8-N3 cAMP was specific since other

SDS-resolvable sperm proteins did not photoincorporate the analog. Caput and cauda sperm pro-

tein photoincorporation could be effectively blocked by low levels of cAMP, but not by cGMP,

ATP or GTP. Sperm epididymal maturation coincided with changes in the cAMP-dependent pro-

tein kinase subunits since cauda sperm contained more available Type II than did caput sperm. A

subcellular analysis of cAMP-dependent protein kinase regulatory subunit in head and tail fractions

was done for caput and cauda sperm and demonstrated that the tail fractions showed more photo-

labeling of both Type I and II regulatory subunits than did the head fractions.

INTRODUCTION

It is generally accepted that morphological

and physiological changes necessary for sperm

function occur during epididymal maturation

(Bedford, 1963; Blandau and Rumery, 1964;

Orgebin-Crist, 1969; Calvin and Bedford, 1971;

Olson and Hamilton, 1978; Laufer et al., 1979;

Hammerstedt et al., 1979; Olson and Danzo,

1981; Hoffer et al., 1981). Although no uni-

form mechanism exists describing this control

of epididymal sperm maturation, the cyclic

nucleotide second messenger system offers an

attractive model for study (Hoskins and Casillas,

1975). This model focuses on cyclic AMP

(cAMP)-dependent protein kinases as multimeric

holoenzymes containing 1 of 2 possible regula-

Accepted September 11, 1984.

Received April 5, 1984.

1 Reprint requests.

tory subunits (R) and two catalytic subunits

(C) in the form of R2C2 (Rannels and Corbin,

1980). The regulatory dimer consists of two

homologous subunits, either R1 (Mr49K)

which elutes from DEAE-cellulose columns at

low salt concentrations, or R11 (Mr=55K) which

elutes at higher salt concentrations (Podesta et

al., 1978). In contrast, the catalytic dimer ap-

pears to consist of identical subunits (Mr4OK)

in both holoenzymes (Zoller et a!., 1979). The

accepted mode of action for the enzyme

proposes that the R subunits bind cAMP,

releasing and thereby activating the C subunits,

which enzymatically transfer the phosphate of

ATP to a protein substrate. These phosphory-

lated proteins, in turn, are considered to be the

means by which cAMP regulates various cell

functions (Greengard, 1978; Krebs and Beavo,

1979; Hoyer et al., 1980). A review of sperm

physiology indicates that such a cyclic nucleo-

tide model may be applicable to the regulation

156 ATHERTON ET AL.

of numerous sperm functions (Garbers and

Kopf, 1980).

An important consideration when evaluating

the interaction between cAMP and sperm

protein is whether or not cAMP itself is acting

as either a first and/or second messenger, or

both, in the mammalian reproductive system

(Hoskins and Casillas, 1975). For cAMP to

work as a first messenger, it would be likely

that a certain fraction of the cell cAMP binding

proteins would be immediately available to

externally added (32P] 8-N3 cAMP. In support

of a cAMP first-messenger model, Gray et al.

(1976) reported that cAMP exists in physi-

ological titers in human semen and seminal

plasma, and Beck et a!. (1975) reported that

extracellular cAMP titers rise significantly in

human cervical mucus at ovulation. Such

extracellular cAMP could be hypothesized to

activate the cAMP-dependent protein kinases

found externally on washed human (Schoff et

al., 1982) and rat sperm (Majumder, 1978;

Forrester et al., 1980; Horowitz et a!., 1981).

In support of this, a recent report showed that

rat vaginal fluid contains cAMP in pmole

concentrations, sufficient to compete with

(32P)8-N3cAMP binding to both RI and RII

regulatory subunits on washed cauda rat sperm

(Atherton et al., 1983). Cyclic AMP undoubt-

edly works as a second messenger in some

sperm cells. This is supported by the observa-

tion that when fucose-containing polysaccha-

rides are released from sea urchin eggs there is

an increase in the rate of sperm Ca� accumula-

tion, stimulation of adenyl cyclase, an increase

in cellular cAMP concentration, and induction

of the acrosome reaction (Kopf and Garbers,

1980). When inhibitors of cyclic nucleotide

phosphodiesterase, the enzyme responsible for

cAMP turnover, are incubated with sperm from

bull (Garbers et al., 1971a,b; Cascieri et al.,

1976), guineapig(Frenkelet al., 1973), hamster

(Morton et al., 1974), human (Homonnai et al.,

1976) and rat (Wyker and Howards, 1977; Paz

et al., 1978), increased sperm motility is

observed that may be attributed to increased

intracellular cAMP concentrations. It has been

suggested, however, that caffeine may stimulate

sperm motility by an as yet unknown mecha-

nism other than a simple inhibition of phos-

phodiesterase (Tamblyn and First, 1977; Levin

eta!., 1981).

Finally, the identification of a phosphory-

lated protein has been correlated to the acquisi-

tion of bull sperm motility (Brandt and Hoskins,

1980; Acott and Hoskins, 1983) and cAMP

concentrations have been reported to increase

in ram epididymal sperm in a caput-to-distal

cauda gradient with an apparent threshold of

cAMP necessary for the initiation of sperm

motility (Amann et al., 1982).

In view of these data, we have used a photo�

affinity analog of cAMP, [32P1 8-N3cAMP, to

study rat sperm cAMP binding. In the presence

of activating wavelengths of light, the azido

group on the analog is converted into a nitrene

which forms a covalent bond to the nearest

amino acid residue (Haley, 1975, 1983; Potter

and Haley, 1982; Khatoon et al., 1983). Conse-

quently, by the discriminatory use of [32P] 8-

N3 cAMP as a site-directed reagent, it is possible

to study certain changes relative to the protein

kinase regulatory subunits.

Previous studies from this laboratory with

[�P] 8-N3 cAMP have shown that: 1) ejaculated

human sperm contain both R1 and R11 cAMP-

dependent protein kinase regulatory subunits;

2) the 8-azido analog is an excellent mimic of

native cAMP in the stimulation of sperm

cAMP-dependent protein phosphorylation; 3)

the analog binding is highly specific (Schoff et

al., 1982); and 4) part of the rat (Forrester et

al., 1980) and human sperm (Schoff et al.,

1982) enzyme activity is externally located.

Given these data and those of other who have

contributed to the description of the sperm

cAMP receptor (Hoskins et al., 1972; Garbers et

al., 1973; Rosado et al., 1976; Lee and Iverson,

1976; Fabbro et a!., 1982; Conti et al., 1983),

the present study was designed to: 1) biochem-

ically describe rat sperm cAMP-dependent

protein kinase regulatory subunits and their

membrane sidedness, 2) determine if changes

occur with the regulatory subunit during

epididymal maturation and 3) examine if there

is a specific subcellular distribution of the

regulatory subunits in the head and tail-midpiece

fraction of caput and cauda sperm. A prelimi-

nary account of this research has been pre-

viously presented (Khatoon, 1983).

Materials

MATERIALS AND METHODS

Nucleotides were purchased from Sigma (St. Louis,MO). [“P1 8-N3 cAMP was synthesized as previouslyreported (Haley, 1975).

Sperm Extraction

Mature albino rats (Holtzman) were anesthetizedwith chloroform and killed by cervical dislocation.

RESULTS

Identification of Subunits

Use of nucleotide photoaffinity probes to

identify specific proteins in impure prepara-

tions must fulfill certain criteria (Potter and

Haley, 1982). Of these, saturation effects near

the known Kd of the photoprobes for the

protein is necessary, and the effects of

various added nucleotides on photoincorpora-

tion should agree with the known effects of the

nucleotides on reversible binding of the photo-

probe. Also, if purified protein standards are

available, they should photolabel and comigrate

on SDS-PAGE identically with the cellular

proteins under study. Figures 1A and B are

autoradiographs of SDS-PAGE on which

proteins of intact caput and cauda sperm cells

were separated after photolabeling with in-

creasing concentrations of F32 Pj 8-N3cAMP.

Both the 49,000- and 55,OOO�Mr subunits

appeared to be saturated at concentrations

between 75-100 nM in agreement with the

known Kd values of 8-N3 cAMP for the regula-

tory subunits of the cAMP-dependent Type I

and Type 11 protein kinase, R1 and R11, respec-

tively. Caput sperm showed only minor incor-

poration in the 55,OOO�Mr subunit and cauda

sperm did not show any significant proteolysis

product at Mr4O,000 ± 10%.

RAT SPERM cAMP BINDING PROTEINS 157

The epididymides were excised from 3 to 6 rats and

trimmed to remove blood vessels and nonseminiferous

tubule tissue. Caput and cauda epididymidal tubuleswere removed by careful dissection and put into ST

buffer (sucrose, 340 mM; Tris, 5mM; pH 7.4; 1 ml/

epididymis) at 20#{176}C.Tubules were minced and sperm

dispersed by mixing on a rotary shaker for 30 mm(setting #6, New Brunswick Rotary Shaker, G76). The

suspension was then filtered through four layers of

cheesecloth and centrifuged at 620 X g for 30 mm at

20#{176}C.The supernatant fraction was discarded and the

sperm resuspended in the original volume of ST andwashed twice at 620 X g for 15 mm at 20#{176}C.The finalpellet was resuspended in a volume of ST to give

approximately 2 X 1O� sperm/mi.

Sperm Fractionation

Sperm were sonicated at 4#{176}Cfor three 30-secperiods using a Bronwill Biosonik Sonicator (2 mm

diameter probe, power 70, tune plus 4) with a 30-sec

4#{176}Ccooling interval between each sonication period.Aliquots of the sonicated sperm homogenates wereused for protein assays (Bradford, 1976). Sperm headand tail fractions were obtained by the procedure of

Calvin (1979). This procedure requires sonication ofsperm until an acceptable separation of heads and tailsis achieved by visual inspection (Fig. 8). When analyzedmicroscopically, cauda sperm are approximately 90%

pure, while caput sperm are slightly less pure (70-

80%). The impurities appear due to erythrocytes and

other nongerm cells. A final sperm concentration of 4

to 8 X 106 sperm/mI is then applied to a differentialstepwise gradient consisting of 13 ml each of 2.2 M;

2.05 M; and 1.8 M sucrose. Centrifugation was done ina Beckman swinging bucket rotor (SW 27) for 1 to 2 h

at 91,000 X g. This procedure resulted in high yields

of 99% pure heads (pellet) and 70-80% tail/midpiece

(1.80/2.05 M interface) fractions, Intact cells were

found at the 2.05/2.20 M interface.Hypotonic rupture of the sperm was accomplished

by resuspending one volume of sperm into 5 vol-

umes of 10 mM phosphate buffer (K2HPO4, 5 mM;

KH2PO4, 5 mM, p1-I 7.5). Freeze fracturing of spermwas completed by 3 cycles of flash freezing sperm in a

slurry of dry ice and isopropyl alcohol followed with

thawing at 4#{176}C.

Photolabeling

Sperm protein (60-80 pg) in KRP buffer (KrebsRinger phosphate; 120mM NaCI, 16mM NaFI2PO4, S

mM KCI, 0.4 mM CaCl2, 1 mM KH2PO4; and 1 mM

MgSO4; pH 7.4) was incubated in planchets with a

final reaction volume of 100 �tl with [32P] 8-N3 cAMP

at 4#{176}Cfor 5 mm. The solution was then photolysed

for 10 mm using a UVS-54 mineral lamp at a distance

of 1.0cm (Haley, 1975). Solutions were intermittentlymixed with a fine jet of air. Photolabeled sperm solu-

tions were then boiled in sealed tubes for 5 mm with a

protein solubilizingmedia(1 mg/mI protein) containing

sucrose, 25%; pyronin Y, 2.5 mg%; sodium dodecyl

sulfate (SDS), 2.5%; dithiothreitol, 23 mg/mI; 13-mer-

captoethanol, 40 �il/ml; EDTA, 2.5 mM and Tris 25

mM at pH 8.0.

Specificity of nucleotide binding was studied by

incubating 80 �g of sperm protein with varyingconcentrations, as indicated, of one of the following

nucleotides: cAMP, cGMP, ATP, or GTP, in a final

volume of 100 M’ of ST buffer at 4#{176}Cfor 10 mm. The

solution was then transferred to another planchetcontaining [32 P1 8-N3 cAMP and incubated at 4#{176}Cfor

10 mm, followed by a 5-mm photolysis and then

solubilization. The final concentration of 1”PI 8-

N3 cAMP was 100 nM. All experiments were repeated

4 or more times with analog from two or more differ-

ent syntheses.

SDS-PA GE Electrophoresis

A 10% to 13.5% linear gradient polyacrylamideslab gel system was used as previously described(Schoff et al., 1982). Molecular weights of � P18-N3 cAMP photolabeled protein were estimated by

comparison of a log molecular weight versus migration

distance plot of commercial protein standards (Owens

and Haley, 1976).

Densicometry and Liquid Scintillation Counting

Autoradiographs were scanned by a densitometer(Ortec model 4310). Photoincorporation was quanti-

fied by laying the autoradiograph over the dried

SDS-gel and cutting out areas of photoincorporation.

Radioactive incorporation was measured with a

Beckman 9000 LS counter using 3.0-mI Beckman

minivials with 3.0 ml of cocktail (5.0 g of PPO and

0.5 g of POPOP/1 of scintillation grade toluene).

A) RAT CAPUT SPERM

55K-

49K---55K

-49K

,.�

I-

-j

-j0

100 200

B) RAT CAUDA SPERM

(C32P8.N3CAMP)

158 ATHERTON ET AL.

nM

55K-?

49K-�

5 101525 5075

-55K

‘-49K

900

800

600

400

200

E

&

�TISIM25 50 75 100 25 150 75 200

(32p38.N3CAMP) nM

FIG. 2. Saturation curves for 132 P1 8-N3 cAMP

photomncorporation into caput and cauda rat spermproteins. Bands of radioactive incorporation weresliced from the dried gel which was used to make the

autoradiograph in Fig. 1 and the radioactive incor-

poration was quantified by liquid scintillation counting.Data were plotted as cpm incorporated at each concen-

tration. Experimental protocol was the same as de-

scribed in Fig. 1.

RAT SPERM cAMP BINDING PROTEINS 159

FIGS. l#{192}and lB. Autoradiograph of an SDS-PAGE on which proteins of rat caput (A) and cauda (B) spermhave been separated after being photolabeled with increasing concentrations of [32 P] 8-N3 cAMP as indicated on

the figure. Photolabeiing, sonication, SDS-PAGE and autoradiography were done as given in Materials and

Methods.

Data presented in Fig. 2 are a quantitative

evaluation of the experiment described in Fig.

1, where bands of radioactive incorporation

were sliced from the gel and quantified

by liquid scintillation counting. These data ful-

filled one of the major criteria of an active site-

directed probe in that they demonstrated satura-

tion effects at the appropriate concentrations.

Another criterion for identification of a

specific binding protein using a photoaffinity

probe is the selective protection by the natural

compound with other compounds, giving little

or no decrease in photoincorporation. Thus, the

effects of cAMP and other nucleotides on

photoincorporation of [32P) 8-N3 cAMP were

studied. The autoradiograph in Fig. 3 shows the

results of one such experiment. Rat caput epi-

didymidal cells were used in this experiment.

Lane 1 shows the incorporation in the absence

of any other nucleotides. Lane 2 shows complete

protection by 100 JIM cAMP and Lanes 3-5,

containing 100 .zM cGMP, 1 mM ATP and 1

mM GTP, respectively, show varying degrees of

photoincorporation. Cyclic GMP has weak

affinity for R1 and R11 and only slightly de-

creased photoincorporation when present at

100 times the photoprobe concentration. ATP

decreased photolabeling in R1 and RII and had

little effect on RII-P04, as expected (Hoyer et

a!., 1980). GTP had essentially no effect.

Figure 4 is an autoradiograph of a SDS-

PAGE on which epididymal sperm proteins and

R1 and R11 standards were separated after

photolabeling with [�P] 8-N3 cAMP. Lanes 1

and 4 contained caput and cauda sperm cell

protein, respectively. Lanes 2 and 3 contained

partially purified preparations of RI and R11

which were run in parallel to aid in the identifi-

cation of the photolabeled sperm proteins. The

three sperm proteins which incorporated over

95% of the [�P1 8-N3 cAMP displayed Mr

values of 55,000 ± 10%, 49,000 ± 10% and

40,000 ± 10%. The two larger Mr values co-

migrated with R11 and R1. The 40,000 ± 10%

species comigrated with a known proteolytic

product of the regulatory subunits (Potter and

Taylor, 1980). This 4O,OOO�Mr species was

rarely observed in cauda cells but was often

observed in caput cells which seemed to have

more endogenous proteolytic activity.

Based on the data shown in Figs. 1 through

4, we concluded that both sonicated caput

and cauda contained primarily R1 and R11

regulatory subunits as the binding sites for

�32 P1 8-N3 cAMP.

Effects of Sperm Cell Disruption on�32 P18-N3 cAMP Photoincorp oration

One of the major questions concerning

sperm cAMP-dependent protein kinases is their

membrane sidedness or their immediate avail-

-68

C.,�30

160 ATHERTON ET AL.

I 2345iii

-92.5 �

m-J

Li�0

�

4�4OC) C)

FIG. 3. Autoradiograph showing the effects of added nucleotides on [32P1 8-N3 cAMP photoincorporation

into rat caput sperm proteins. Sonicated rat caput sperm equivalent to 60 �ig of protein were incubated in KRP

buffer, pH 7.4, at 4#{176}Cfor 5 mm with 100 nM [“P18-N3 cAMP and the indicated nucleotides prior to photo-lysis. Cyclic AMP (Lane 2) and cGMP (Lane 3) were present at 100 pM concentration, whereas ATP (Lane 4)

and GTP (Lane 5) were at 1 mM concentration. After 10 mm of photolysis as described earlier, reaction was

stopped by adding PSM. SDS-PAGE and autoradiography were. done as described in the text.

55K�I

49K- 49K#{149}

� 40K- 40K

0

FIG. 4. Autoradiograph of an SDS-PAGE on which [“P18-N3 cAMP-photolabeled partially purified Rj and

R11 subunits and rat caput and cauda sperm proteins were separated. Rat caput and cauda sperm, equivalent to

70 pg of protein, were added to Lanes 1 and 4, respectively. Lanes 2 and 3 contained the partially purified RI

and R11 preparation. Photolabeling was done at 100 pM [32 PJ 8-N3 cAMP. Procedures for SDS-PAGE and auto-

radiography were as described in Materials and Methods.

49�00

bx

E0.

0LU

a::0U-

00

a-

C-,

za,I-sa-

CsJ

5

0 CAPUT

� CAUDA

3�

2

4O�00

ftrL JL IWC FT SN HS WC FT SN HS WCFT SN HS

RAT SPERM cAMP BINDING PROTEINS 161

ability to externally added cAMP or cAMP

analogs. To investigate this problem, intact

sperm cells were subjected to the procedures of

freeze thawing, sonication and hypotonic

rupture, and the effects of these perturbations

on [32P18-N3cAMP photoincorporation deter-

mined. In that experiment, sperm cells in

epididymal fluid were washed as described in

Materials and Methods. This procedure resulted

in intact cells free of seminal plasma, and

constituted the whole cell fractions. By conven-

tion, over 85% of this cell population excluded

0.5% eosin, indicating they were primarily

intact cells (Eliasson, 1977). This observation

also was supported by visual estimation of cell

motility (Schoff, 1981). Aliquots from this sus-

pension were then disrupted either by freeze-

thawing, sonication, or hypotonic treatment as

described in Materials and Methods. Portions of

these four samples were used for photolabeling

experiments with [32 P1 8-N3 cAMP to determine

the level of photoincorporation of the analog.

6

CELL TREATMENTS

55.000

FIG. 5. Effect of various perturbations on [32 P1 8-N3 cAMP photoincorporation into rat epididymal sperm

proteins. Rat epididymal sperm were extracted by standard procedures described in the text. Aliquots containing

80 pg protein from whole cells (WC), frozen-thawed cells (Fr), sonicated cells (SN) or hypotonically rupturedcells (HS) were incubated in a total volume of 100 p1 in KRP buffer. The reaction mixture also contained 250nM [32 P1 8-N3 cAMP. After 5 min incubation at 4#{176}C,it was photolysed for 10 mm at the incubation temperature

using a UVS-54 Mineralite lamp at a distance of 1 cm. Photolabeled sperm solutions were solubilized by adding

PSM, and proteins were separated by SDS-PAGE. Electrophoresis, autoradiography and quantification of 132 P18-

N3 cAMP incorporation were done as described in Materials and Methods. Mr values of labeled proteins areshown above bars on the graph.

CAPUT (R1 .R1)

4 6

NUMBER OF 30 SECOND SONICATIONS

162 ATHERTON ET AL.

Figure 5 shows the data plotted from such an

experiment. It reveals that sonication appeared

to be the best physical method for exposing the

most cAMP binding proteins in both types of

epididymal sperm. Two major protein bands

were photolabeled in both sources of sperm.

The molecular weight, expressed as an Mr value,

corresponded to those of R1 and R11 subunits

of protein kinase, i.e., 49,000 ± 10% Mr and

55,000 ± 10% Mr, respectively. Incorporation

was also observed in protein with a Mr4O,000,

primarily in caput sperm, which was shown to

be a proteolysis product of R1 and R11. In

comparison to intact cells, sonication ex-

posed about 59% and 119% more R1 in caput

and cauda epididymidal sperm, respectively.

Similar treatment exposed more than 100% and

140% R11 in caput and cauda sperm, respective-

ly. Hypotonic treatment of cells was not as

effective as sonication and usually resulted in

very little increase in photoincorporation into

R11. Freeze thawing did not appear to expose

additional sites at all.

Because sonication appeared the most

effective procedure for exposure of RI and R11,

further experimentation was done to evaluate

the degree of sonication which would expose

the maximum number of receptors. Figure 6

demonstrates the results of such an experiment.

Intact washed sperm were subjected to an

increasing number of 30-sec sonications,

separated by 30-sec intervals. Aliquots from

each time point were removed and photolabeled

with 12P18-N3cAMP. Proteins were separated

by SDS-PAGE and bands containing radioactive

incorporation on the dried gel were identified

by autoradiography. These were sliced from the

gel and quantified by liquid scintillation count-

ing. The data show that the total incorporation

of the analog in caput and cauda sperm increased

with the degree of sonication, with approxi-

mately four 30-sec sonications giving maximum

values. Further sonication resulted in decreased

labeling, either due to protein denaturation or

the release of endogeneous, compartmentalized,

cAMP which competed for the binding sites.

The latter was supported by the lack of a

significant decrease in photoincorporation after

eight 30-sec treatments. This seemed to be true

with both types of sperm. On a scale of 100%

incorporation at the peak in both types of

sperm, about 65% of the cAMP binding proteins

appeared not accessible in intact cell prepara-

tions that were at least 85% intact and motile.

These were presumed to be intracellular proteins.

Photolabeling of Sperm in Epididymal Fluid

To ensure that our washing procedure was

not disrupting sperm, the cells were gently

extracted from the cauda epididymidis and im-

mediately photolabeled with [32P1 8-N3 cAMP

in epididymal fluid, without centrifugation, and

an aliquot prepared for electrophoresis. These

photolabeled cells were then washed three

times using the wash procedure (Materials and

Methods), with aliquots of each supernatant

and pellet prepared for electrophoresis (Fig.

7A). For contrast, a similar experiment was

done with photolabeling carried out on separate

aliquots of the initial unwashed sperm and each

supernatant and pellet of the subsequent

washing steps (Fig. 7B). In all samples, both R11

(5 5K) and R1 (49K) Mr subunits were photo-

labeled. More photoincorporation appeared in

N

EC,

FIG. 6. Effects of increased sonication on 132 P18-

N3 cAMP photoincorporation into rat epididymal

sperm proteins. Washed epididymal sperm were

sonically disrupted for the indicated number of times.

Aliquots (equivalent to 60 pg proteins) from each time

point were incubated with 100 nM [32P18-N3cAMP

for 5 mm at 4#{176}C,photolysed for 10 mm using a UVS-

54 lamp at a distance of 1 cm, solubilized, and the

proteins separated by SDS-PAGE. Electrophoresis,autoradiography and quantification of 132 P1 8-N3

cAMP incorporation were done as described in Materi-

als and Methods.

RAT SPERM cAMP BINDING PROTEINS 163

the unwashed sperm and pellets than appeared

in the supernatants in preparations that were

photolabeled either before or after washing.

Microscopic examination of the 620 x g

supernatants revealed a few sperm, which could

account for some of the photoincorporation

seen in the supernatants. However, it appears as

if some of the cAMP binding proteins were

loosely held to the sperm cells and washed

off, or out, rather easily. In both experiments,

it could be observed in the 2nd and 3rd pellets,

and to a lesser extent in their supernatants, that

some proteins of molecular weight of less than

40K Mr were photolabeled. These were identi-

fied as R1 and R11 proteolytic products.

Distribution of Type I and

Type II Regulatory Subunits

in Caput and Cauda Sperm

To quantitatively evaluate the distribution

of [32P1 8-N3 cAMP-photolabeled R1 and R11

subunits of cAMP-dependent protein kinase, a

number of experiments were done on sonicated

sperm cells treated to give maximum photo-

incorporation of [2P] 8-N3 cAMP. Data were

analyzed statistically and are presented in Table

1. Levels of photoincorporation into R1 subunits

were quite similar in both caput and cauda

epididymidal sperm. However, R11 photolabel-

ing was significantly higher (P>0.01) in cauda

sperm than in caput. Caput R1 photoincorpo-

rated more [2P] 8-N3 cAMP than did caput

RII (P>0.007). In cauda cells, [32P]8-N3cAMP

did not show a difference between photoincor-

poration into R1 and R11. As a result, the

R1/R11 in cauda cells was closer to unity, as

compared to an average of 3.72 for caput cells.

Subcellular Distribution

of Regulatory Subunits

Figure 8 presents a typical preparation of

cauda sperm subcellular fractions, obtained

after sonication and discontinuous sucrose

density gradient centrifugation. A shows intact

sperm, and B and C show the separated heads

and tails, respectively. The method appeared to

be appropriate for this type of fractionation,

since about 99% of the heads and 90-95% of

tail/midpiece fractions were found in the

respective fractions. The interface at 2.05

M/2.2 M sucrose contained the intact sperm.

This fraction was discarded.

Subcellular fractions of both caput and

cauda sperm were subjected to photolabeling

with saturating levels of [32P18-N3cAMP.

Figure 9 is an autoradiograph of these fractions

separated on SDS-polyacrylamide gels. It

appeared that head fractions prepared in this

fashion (Lanes 2 and 5) were mostly devoid of

any major amounts of cAMP binding proteins,

or still had these proteins located such that

they are not available to added 8-N3cAMP.

Tail/midpiece fractions (Lanes 3 and 6) seemed

to be the major site of these binding proteins. A

large portion of the caput cAMP binding

proteins appeared to be detached from the

sperm during fractionation technique, whereas

those of cauda sperm appeared to be firmly

attached to the tail/midpiece fraction. In other

experiments using caput cells, less rigorously

sonicated, larger amounts of R1 and R11 were

observed in tail/midpiece sections.

Table 2 summarizes the statistical analysis

of [32P] 8-N3 cAMP photoincorporation into

cAMP-dependent protein kinase regulatory

subunits in epididymal sperm subcellular

fractions. A comparison of sonicated sperm of

caput and cauda origin readily shows the

consistency of the data with that of Table 1.

The mean cpm values incorporated into R1

and R11 were significantly different from one

another in caput sonicated cells and tail/mid-

piece subcellular fractions (P< 0.001). However,

the corresponding values in cauda sonicates and

tail/midpiece subcellular fractions showed little,

if any, significant variation. It should be noted

that the distribution of R1 in the subcellular

fractions of caput and cauda cell types was

significantly different among these cell types,

P<O.001 and <0.007 in heads and tails, respec-

tively, although R1 had a similar mean value in

sonicated caput and cauda cells. This result was

attributed to the loss through detachment, or

solubilization, of caput R1 and R11 in the frac-

tionation procedures.

DISCUSSION

The purpose of the research presented herein

was to identify the cAMP binding proteins of

both caput and cauda rat sperm, and determine

the membrane sidedness and cellular location of

these proteins. The differences between caput

and cauda sperm cells may be viewed as matu-

rational changes that occur in the epididymis.

The results in Figs. 1 through 4 show that

both caput and cauda epididymidal sperm con-

tain both the Type I and Type II regulatory

subunits of cAMP-dependent protein kinase (R1

CEF EF C WS1 WC1 WS2 WC2

164 ATHERTON ET AL.

CEF EF C - WS1 WC1 WS2 WC2

A) RAT CAUDA

55K-i

d 49K-i

55K-

d 49K-’

B) RAT CAUDA

SPERM LABELED AND THEN WASHED

SPERM WASHED AND THEN LABELED

RAT SPERM cAMP BINDING PROTEINS 165

TABLE 1. Distribution of [32P1 8-N, cAMP-photolabeled Type I and Type II regulatory subunits of cAMP-depen-

dent protein kinase in rat epididymal sperm.a

Subunits Caput Cauda Probability

R1R11

47.86 ± 10.7116.48 ± 6,56b

82.43 � 26.o3’94.46 ± 34.40

NSP>O.O1

R1+RII 64.34 ± 15.08 176.89 ± 60.07 P<0.03R1/R11 3.72 ± 0.78 0.94 ± 0.095 P>0.004

aSperm were photolabeled as described in Materials and Methods. Values represent the mean ± SEM of the

area (mm2) under each peak in the densitometer tracings of the autoradiographs; n10 in each case.

bcaput R1>R11 (P>0.007).

cCauda Rj is not significantly different from cauda R11. Autoradiographic data was verified by liquid scintilla-

tion counting (data not shown).

and R11, respectively). These results agree with

our earlier reports (Schoff et al., 1979; Forrester

et al., 1980; Khatoon et al., 1981), as well as

those of others (Horowitz et al., 1981; Fabbro

et al., 1982; and Conti et al., 1983), that

mammalian sperm contain both Types I and II

cAMP-dependent protein kinases. Proof that

the photolabeled proteins discussed in this

paper are R1 and R11 subunits is as follows.

First, 8-N3 cAMP is an excellent biological

mimic of cAMP and activates the Types I and 11

protein kinases maximally at 75 nM to 125 nM

concentrations (Owens and Haley, 1976).Figures 1 and 2 show that [2P)8-N3cAMP

saturates the photolabeled species within this

concentration range. That these are R1 and R11

subunits is also supported by their Mr values

of 49K and 55K, respectively, and the observa-

tion shown in Fig. 4 that the photolabeled

species also comigrates with a partially purified

preparation of porcine heart R1 and R11.

Further confirmation is provided by Figure

3 which shows that cAMP was the only nu-

cleotide that was effective at preventing [32P] 8-

N3 cAMP photoincorporation. Support that the

doublet at 55K is R11 in the phosphorylated

(R11-PO4, highest Mr) and dephosphorylated

forms (RII, lowest Mr) is shown in Lane 4

where ATP is present. Here the R11 subunit

disappears as it is converted to R11-P04, as

expected (Hoyer et al., 1980). Other than this,

none of the nucleotides seem to affect �32 P1 8-

N3 cAMP photoincorporation into R11/R11-P04

except cAMP. Both cGMP and ATP at concen-

trations much greater than [32P1 8-N3 cAMP

decrease photoincorporation into R1, but to an

extent much less than cAMP. Cyclic GMP isknown to have a low affinity for the cAMP

site(s) and this probably explains its effect.

ATP is known to decrease the affinity of RI for

cAMP and 8-N3cAMP (Haddox et al., 1972;

Beavo et al., 1974; Owens and Haley, 1978);

therefore, a slight decrease in labeling of R1 in

the presence of ATP would be expected.

An interesting and unusual observation made

in this experiment was the separation of R1 into

two closely migrating species. Since R1 is

thought to be a phosphorylated species, it is

possible that this separation reflects different

phosphorylated forms of R1. Also, other

isoprotein forms of minor proteolysis may be

the cause. However, cGMP and ATP appear to

have different effects on photoincorporation

into this doublet, with ATP not preventing

FIG. 7. A) Photolabeling of unwashed cauda sperm in epididymal fluid and the effects of subsequent washes

on [32 P1 8-N3 cAMP-photolabeled proteins. Cauda sperm in epididymal fluid (C-EF) was photolabeled and a por-tion of the sperm-fluid mix was subjected to SDS-PAGE and autoradiography (Lane 1). Photolabeled cauda

sperm was then separated from epididymal fluid (EF) by centrifugation and a portion of the fluid (EF, Lane 2)

and sperm cells (C, Lane 3) treated as above. The same sperm cells were washed two times by centrifugation inKRP buffer with Lanes 4 and 6 containing portions of the supernatant of the first (WS) and second wash (WS,),

respectively. Lanes 5 and 7 contained first (WC1) and second (WC2) washed sperm cells. B) Same conditions as

above except each fraction was photolabeled after the separation of the supernatant and pelleted sperm cells ineach subsequent centrifugation step. All experiments were done at room (20#{176}C)temperature.

166 ATHERTON ET AL

FIG. 8. Photograph of rat cauda epididymidal sperm and its subcellular fractions. Epididymal sperm were sub-

jected to sonicazion to separate the heads from tail/midpiece fractions, and were then fractionated by discontin-

uous sucrose density gradient centrifugation. Aliquots from these fractions were examined on a phase-contrastmicroscope and photographed at 40X. A) Intact sperm; B) sperm heads; and C) sperm tail/midpiece.

photomncorporation into the faster migrating

species as well as cGMP.

The experiments whose results are shown in

Figs. 5-7 were designed to observe the mem-

brane sidedness of Rj and Rh. This was done to

test earlier reported observations that rat

(Majumder, 1978; Forrester et al., 1980; and

Schoff et al., 1982) and human sperm (Rosado

et al., 1977; Schoff et al., 1982) contain an

externally accessible cAMP-dependent protein

kinase. The possible presence of such an exter-

nally located kinase is of interest since cAMP

CpS cpH CPTCdS CdH CdT

RAT SPERM cAMP BINDING PROTEINS 167

I 2 3 4 5 6

FIG. 9. The distribution of [32 P1 8-N3 cAMP binding proteins between head and tail/midpiece fractions. Rat

caput and cauda sperm were sonicated to fracture heads into tail/midpiece sections. These were purified by den-

sity centrifugation as given for Fig. 8. Photolabeling at 300mM 132 P1 8-N3 cAMP was done on original caput andcauda sonicates (Lanes 1 and 4, respectively), caput heads (Lane 2) and tail/midpiece (Lane 3), cauda heads(Lane 5) and tail/midpiece (Lane 6). SDS-PAGE and autoradiography were done as given in Fig. 1.

has been found in human cervical mucus at the

time of ovulation (Beck et al., 1975) and in rat

vaginal fluids during estrus and early metestrus

(Atherton et al., 1983). Also, the latter study

demonstrated that ovariectomy reduced vaginal

fluid cAMP titers to background levels. The

possible importance of these studies concurs

with the work of Pariset et al. (1983) who

demonstrated a positive correlation between

cAMP-dependent ect.okinase and human sperm

motility, and Perreault and Rogers (1982) who

showed that cAMP reduces the time required

for in vitro capacitation to occur.

The set of experiments shown in Fig. 5 were

done primarily to determine which type of cell

disruption technique exposed the maximum

number of cAMP binding sites. Considering

this, sonication was the most effective proce-

dure, followed by hypotonic rupture. Our

method of freeze thawing did not appear to

increase availability above that available in

intact cells. Another observation, consistent

with the data in the autoradiograms of Figs. 1,

3, 4 and 9, is that caput sperm always seem to

have a much larger amount of the 40K�Mr

photolabeled species present than do cauda

sperm. This is the result of endogenous prote-

olysis and was always present even in intact

caput cells.

The data in Fig. 6 show the effects of in-

creased sonication of E32P1 8-N3 cAMP photo-

incorporation into total R1 plus R11 binding

proteins of caput and cauda sperm. Through

several experiments, it was routinely observed

that more cAMP binding proteins were available

for photolabeling in cauda sperm compared to

caput sperm. Also, approximately 37% of cauda

and caput sperm cAMP binding proteins

appeared to be externally available in nonsoni-

cated cells. Routinely, these cells showed over

85% viability after three washes, indicating that

the externally available binding proteins were

not due to disrupted cells. However, these

results, as well as the earlier results of Schoff et

al. (1982), are in sharp contrast to the report

by Horowitz et al. (1984) which indicates that

all cAMP binding proteins are available to ex-

ternally added 132 P1 8-N3 cAMP and a-chymo-

trypsin. Our observations suggest that approxi-

mately 60% of the cAMP binding proteins of

168 ATHERTONETAL.

intact sperm require sonication to be readilyN

available to [32P1 8-N3 cAMP or proteolyticN -4 N U

“ - -� C enzymes. We cannot explain this discrepancy.+4 +4 +3 +3 .9

However, the isolation of high-quality intactN �N =

is 0 � � ,.. sperm from epididymal fluid is not trivial. SinceI-’ ‘o -l N C‘0 0 o o a rise in intracellular sperm cAMP levels, as

5, 0 - ‘-4 Nshown by Hoskins et al. (1972) and Garbers

N ‘ ‘ �‘ and Kopf (1980), occurs during epididymal0 0 0 N0 0 0 0 -

migration, it seems most likely that some‘0 V V V V

‘0 membrane sidedness for cyclic nucleotide musta., a. a. a.

exist. Otherwise, relevant measurements of‘0

In -N - .0 changing intracellular concentrations could not

- 0’. have been made. Also, we assayed for hexo--4 -4 -4

41 +3 +3 +1‘I. kinase and our results showed essentially noN release of this enzyme into our buffered washes.

0.,� I- ii� 0 v’. .-

0 0. 0 � To further establish the existence of exter-,,,, 0 In -. ‘5’ CCo n -o nally available sites the sperm cells were photo-

,-�

.9 N labeled at 20#{176}Cin epididymal fluid immedi-

- ately after being gently removed from the,I� In NON ‘� epididymis. Data on sperm cells photolabeled

V -H +3 +3 +4

and then washed are shown in Fig. 7A andNN

0. �, ‘.�� .� contrasted with sperm cells that were washed

E 0 N N * u and then photolabeled as shown in Fig. 7B.“0

Lanes 1, 3, 5 and 7 in both Figs. 7A and B- N ‘ contain the same number of cells. The even-�00In

numbered lanes contain an equal portion of the‘0

V V V 41 supernatant from each centrifugation step. Thea., a. a. a.

results indicate that sperm cells in epididymal0’.

In o 0 ‘�‘ fluid, without being exposed to harsh treatment,In �n ‘5#{149} 0.V

.0 4’ 4’ have readily available cAMP binding proteins.is

Also, at least some of these binding proteins are-4

susceptible to removal by gentle washing pro-,0 �. ‘0 in N

cedures, indicating that they are loosely held to- ‘01,

c the external surface. They quite likely may be.0,9

�‘. , located on cytoplasmic droplets, as proposed-4 � by Tash and Means (1983). However, the bulk0’ .“ � ‘� of the binding proteins remain bound to the

‘0 -H 43 +4 +1

-� cells after removal of the epididymal fluid andv NN

- ‘5. in two subsequent washes (compare Lanes 1 and 7N � In 8.0

0 �n ‘5- - N � E in Figs. 7A and B).

-c.9 Z Table 1 reflects an extensive analysis of the

� “� ‘�‘ ‘� Il differential distribution of R1 and R11+R11-P04U) o u found in caput versus cauda sperm cells where

u Z 0 0 0 U.�

‘I V V Va. a. a., a. cauda routinely showed 3.5 to 4 times more,� photoincorporation into R11. The major differ-

‘-4N .-4 � N ‘� � ences appear to be a very low level of R11 in

- ‘.c; v’� - �‘.4., N _ .� .� caput and, in general, a larger number of Rj and

+4 -H +4 #1C ‘� .� Rj� molecules available to [�P1 8-N3 cAMP in

.90’ 0 cauda cells. This does not mean that cauda con-

In - ‘5’ -4U) N � 41’. ‘0 tains more R1 and R11 molecules, only that

I., 0. 0 In ‘0� 0 - -

more is available to bind the photoprobe. Anexplanation for the low levels of R11 in caput

ns .� - -,,� C - �. .� may be obtained from the observation that in-

� i�’ creased proteolysis of photolabeled species is.4: -� - � beI- U) .9 observed in caput. However, if this is the case,

REFERENCES

Acott, T. S. and Hoskins, D. D. (1983). Cinemato-graphic analysis of bovine epididymal spermmotility: epididymal maturation and forward

motility protein. J. Submicrosc. Cytol. 15:77-82.

Amann, R. P., Hay, S. R. and Hammerstedt, R. H.(1982). Yield, characteristics, motility and cAMP

content of sperm isolated from seven regions of

the ram epididymis. Biol. Reprod. 27:723-73 3.Atherton, R. W., Gem, W., Culver, B., Seitz, J., Veal-

Ward, B. and Khatoon, S. (1983). Rat vaginal

fluid cAMP concentrations and its interaction

with spermatozoa. Biol. Reprod. 28: 145a.

Beavo, J. A., Bechtel, P. J. and Krebs, E. G. (1974).

Preparation of homogeneous cyclic AMP-depen-

dent protein kinasis and its subunits from rabbit

skeletal muscle. Methods Enzymol. 38:299-308.

Beck, K. J., Hungersh#{246}fer, R., Herschel, S. and Sch.dn-h#{246}fer. (1975). Cyclic nucleotide levels in human

semen and cervical mucus; possible function in

reproductive process? In: Proc. IV European

Sterility Congress (Cortes-Preito, J. and K.

Semm, eds.). PubI. cient. Reprod. Hum. y Anim,Madrid, Spain, pp. 45-49.

Bedford, J. M. (1963). Morphological changes inrabbit spermatozoa during passage through the

epididymis. J. Reprod. Fertil. 5:169-177.

Blandau, R. J. and Rumery, R. E. (1964). The relation-ship of swimming movements of epididymal

spermatozoa to their fertilizing capacity. Fertil.

Steril. 15:571-579.Bradford, M. M. (1976). A rapid and sensitive method

for the quantitation of microgram quantities of

RAT SPERM cAMP BINDING PROTEINS 169

the R11 is much more susceptible than R1 in

sperm cells, while such is not the case in other

cell systems (Haley, unpublished observations).

Also, assuming that all of the 40K and 38K

species were from R11 and not R1, then the

total of R11 + the 40K and 38K species still

would not usually equal half the level observed

in R1 (see Figs. 1 and 3). All interpretation of

[32P18-N3 cAMP photoincorporation data, in

comparison to [3H]cAMP binding, should be

done with the knowledge that 8-N3 cAMP is

not a substrate for mammalian cAMP phos-

phodiesterase (Hoyer et al., 1980).

Another explanation would be that R11 is

loaded with “cold-trapped” endogenous cAMP

(Owens and Haley, 1978; Khatoon et al.,

1983), which blocks [�P)8-N3cAMP binding.

However, one must then involve differential

compartmentahization of R1 and R11 since R1

binds cAMP significantly tighter than R11 in

purified form and in every tissue homogenate

(heart, lung, liver, etc.) that we have tested. It is

also possible that R11 is selectively blocked by

some unknown epididymal factor, such as a

protein kinase inhibitor, which prevents cyclic

nucleotide binding. This inhibitor may be lost

during the maturation process where cauda

sperm emerge with R11 unblocked. At present

there is no resolved explanation for this differ-

ence between caput and cauda cells. Also there

is no proof that this difference in photolabeled

Rj and R11 ratios has a major effect on the bio-

logical properties of these cells (e.g., the in-

creased fertilization capacity of cauda cells).

However, we are convinced that these observa-

tions are worthy of further studies and must be

taken technically into account when considering

the effects of cAMP on these cells and mammali-

an reproduction in general.

The subcellular location of the cAMP binding

proteins between head and midpiece/tail

fractions was determined as shown in Figs. 8

and 9. Tails totally free of heads were easy to

prepare, but the head fraction was always

contaminated by small tail fragments (see Fig.

8). This may be the reason for the small amount

of photolabeling seen in the head fractions of

Fig. 9. However, the major observation is that

most, if not all, of the readily available cAMP

binding proteins are located in the midpiece/tail

region, as given in Table 2. These data are not

meant to imply that no Rj or R11 exists in head

fractions. These heads were not significantly

disrupted (see Fig. 8) and the cAMP binding

proteins of the head fraction may not have

been available to the added �32 P1 8-N3 cAMP.

In summary, the results obtained in this

study demonstrate that rat caput and cauda

sperm cells contain both R1 and R11 cAMP-

dependent protein kinases. Also, consistent

with other reports (Majumder, 1978; Schoff et

al., 1979) it appears as if approximately 35 ±

5% of the cAMP regulatory binding proteins are

externally located in both intact unwashed and

washed cells. This observation is also supported

by our current work in preparation which

shows external ATP and GTP binding sites as

well as external protein kinase activity as

followed by phosphorylation of endogenous

protein substrates (see Haley, 1983). In addi-

tion, the location of the majority of the cAMP

binding proteins was localized in the midpiece/

tail section with very minor labeling being

observed in the heads. We feel this implies a

role for cAMP in sperm cell motility since

cAMP levels have been found to fluctuate in

oviduct fluids. However, a definitive role for

the cAMP-dependent protein kinase of mam-

malian sperm cells has not yet been identified.

170 ATHERTON ET AL.

protein utilizing the principle of protein-dye

binding. Anal. Biochem. 72:248-254.

Brandt, H. and Hosk ins, D. D. (1980). A cAMP-depen-

dent phosphorylated motility protein in bovine

epididymal sperm. J. Biol. Chem. 255:982-987.

Calvin, H. I. (1979). Isolation of stable structures fromrat spermatozoa. In: The Spermatozoa: Matura-

tion, Motility, Surface Properties, and Compara-

tive Aspects (D. W. Fawcett and J. M. Bedford,eds.). Urban & Schwarzenberg. Baltimore, pp.

387-391.

Calvin, H. I. and Bedford, .1. M. (1971). Formation ofdisulfide bonds in the nucleus and accessory

structures of mammalian spermatozoa during

maturation in the epididymis. J. Reprod. Fertil.(Suppl.) 13:65-76.

Cascieri, M., Amann, R. P. and Hammerstedt, R. H.

(1976). Adenine nucleotide changes at initiation

of bull sperm motility. J. Biol. Chem. 251:787-

793.

Conti, M., Adamo, S., Geremia, R. and Monesi, V.

(1983). Developmental changes of cyclic adeno-sine monophosphate-dependent kinase activity

during spermatogenesis in the mouse. Biol. Re-

prod. 28:860-869.Eliasson, R. (1977). Supravital staining of human

spermatozoa. Fertil. Steril. 28:1257.Fabbro, D., Jochum, A., Balerna, M., Campana, A. and

Eppenbergem. (1982). Cyclic adenosine mono-

phosphate-dependent protein kinases of human

seminal plasma: Origin and characteristics of

multiple forms. Biol. Reprod. 27:159-169.

Frenkel, G., Petersen, R. N. and Freund, M. (1972).

The role of adenine nucleotides and the effect of

caffeine and dibutyryl cAMP on the metabolismof guinea pig epididymal spermatozoa. Proc. Soc.

Exp. Biol. Med. 144:420-425.

Forrester, I. T., Schoff, P. K., Haley, B. E. and Ather-

ton, R. W. (1980). Determination of protein

kinase activity in intact mammalian sperm. J.

Androl. 1:70-71a.

Garbers, D. L. and Kopf, C. S. (1980). The regulation

of spermatozoa by calcium and cyclic nucleo-

tides. Adv. Cyclic Nucleotide Res. 13:251-307.Garbers, D. L., First, N. L., Sullivan, J. J. and Lardy,

H. A. (1971a). Stimulation and maintenance of

ejaculated bovine spermatozoa respiration andmotility by caffeine. Biol. Reprod. 5:336-339.

Garbers, D. L., Lust, W. D., First, N. L. and Lardy, H.

A. (1971b). Effects of phosphodiesterase inhibi-

tors and cyclic nucleotides on sperm respiration

and motility. Biochemistry 10:1825-1831.Garbers, D. L., First, N. L. and Lardy, H. A. (1973).

Properties of adenosine 3’,S’-monophosphate-

dependent protein kinases isolated from bovine

epididymal spermatozoa. J. Biol. Chem. 248:

875-879.Gray, J. P., Drummond, G. I., Luk, D.W.T., Hardman,

J. G. and Sutherland, E. W. (1976). Enzymes of

cyclic nucleotide metabolism in invertebrate and

vertebrate sperm. Arch. Biochem. Biophys.

172: 20-30.

Greengard, P. (1978). Phosphorylated proteins as

physiological effectors. Science 199:146-152.

Haddox, M. K., Newton, N. E., Harth, D. K. and

Goldberg, N. D. (1972). ATP (Mg2+) inducedinhibition of cyclic AMP reactivity with a skeletal

muscle protein kinase. Biochem. Biophys. Res.Commun. 47:653-661.

Haley, B. (1975). Photoaffinity labeling of cAMP

binding sites of human red blood cell membranes.

Biochemistry 14: 3852-3857.Haley, B. (1983). Development and utilization of

nucleotide photoaffinity probes. Fed. Proc.

42:2831-2836.

Hammerstedt, R. H., Keith, A. D., Hay, S., Deluca, N.,

and Amann, R. (1979). Changes in ram sperm

membranes during epididymal transit. Arch.

Biochem. Biophys. 196:7-17.

Hoffer, A. P., Shaleu, M. and Frisch, D. H. (1981).

Ultrastructure and maturational changes inspermatozoa in the epididymis of the pigtailed

monkey, Macaca uesesrrina. Am. Soc. Androl.3:140-146.

Homonnai, Z. T., Paz, G., Sofer, A., Kraicer, P. F. and

Havell, A. (1976). Effect of caffeine on themotility, viability, oxygen consumption and

glycolytic rate of ejaculated human normodinetic

and hypokinetic spermatozoa. Int. J. Fertit.

21: 163-170.

Horowitz, J. A., Toeg, H. and Orr, G. A. (1981).

cAMP-dependent protein kinases on the outer

surface of rat caudal epididymal sperm. J. Cell

Biol. 91:189a.Horowitz, J. A., Toeg, H. and On, G. A. (1984).

Characterization and localization of cAMP-

dependent protein kinases in rat caudal epididy-

mal sperm. J. Biol. Chem. 259:832-838.

Hoskins, D. D. and Casillas, E. R. (1975). Function of

cyclic nucleotides in mammalian spermatozoa.In: Handbook of Physiology, sec. 7, vol. 5 (R. 0.

Greep and E. B. Estwood, eds.). AmericanPhysiological Society, Bethesda, MD, pp. 453-

460.Hoskins, D. 0., Casillas, E. R. and Stephens, D. T.

(1972). Cyclic AMP-dependent protein kinases of

bovine epididymal spermatozoa. Biochem. Bio-

phys. Res. Commun. 48:1331-1338.

Hoyer, P., Owens, J. R. and Haley, B. (1980). Use ofnucleotide photoprobes to elucidate molecular

mechanisms of nucleotide regulated phenomena.

Ann. N.Y. Acad. Sci. 346:280-301.

Khatoon, S. (1983). A study of sperm cell phosphory-

lating systems using nucleotide photoaffinity

probes. PhD Thesis. University of Wyoming,

Laramie, WY 82071.

Khatoon, S., Schoff, P. K., Haley, B. E. and Atherton,

R. W. (1981). A comparative study of sperm

cAMP-dependent protein kinases by a photo-affinity analysis. J. Androl. 2:35a.

Khatoon, S., Atherton, R. W., Al-Jumaily, W. and

Haley, B. E. (1983). Use of nucleotide photo-

affinity probes to study hormone action. Biol.

Reprod. 28:61-73.

Kopf, G. S. and Garbers, D. 0. (1980). Calcium and a

frucose-sulfate-rich polymer regulate sperm cyclicnucleotide metabolism and the acrosome reaction.

Biol. Reprod. 22:118-1127.

Krebs, E. G. and Beavo, J. A. (1979). Phosphorylation-

dephosphorylation of enzymes. Annu. Rev. Bio-

chem. 48:923-959.Laufer, N., May, J. V., Segal, S. and Grover, N. B.

(1979). Size of rat spermatozoa during matura-

tion along the epididymis. Arch. Androl. 3:293-

RAT SPERM cAMP BINDING PROTEINS 171

299.

Lee, M.Y.W. and Iverson, R. M. (1976). An adenosine

34, 5’-monophosphate-dependent protein kinase

from sea urchin spermatozoa. Biochem. Biophys.

Acta 429:123-126.

Levin, R. M., Greenberg, S. H. and Wein, A. J. (1981).

Quantitative analysis of the effects of caffeine on

sperm motility and cyclic 3’,5’-AMP phosphodi-

esterase. Fertil. Steril. 36:798-802.Majumder, G. C. (1978). Occurance of a cyclic AMP-

dependent protein kinase on the outer surface of

rat epididymal spermatozoa. Biochem. Biophys.

Res. Commun. 83:829-836.

Morton, B., Harrigan-Lum, J., Albagli, L. and Jooss, T.

(1974). The activation of motility in quiescenthamster sperm from the epididymis by calcium

and cyclic nucleotides. Biochem. Biophys. Res.

Commun. 56:372-379.

Olson, G. E. and Danzo, B. J. (1981). Surface changes

in rat spermatozoa during epididymal transit.

Biol. Reprod. 24: 43 1-443.

Olson, C. E. and Hamilton, D. W. (1978). Characteriza-

tion of the surface glycoproteins of rat spermato-

zoa. Biol. Reprod. 19:26-35.Orgebin-Crist, M. C. (1969). Studies on the function

of the epididymis. Biol. Reprod. 1:155-175.

Owens, J. R. and Haley, B. E. (1976). A study of

adenosine-3’, 5’-cyclic monophosphate binding

sites on human erythrocyte membranes using

8-azido-adenosine-3 ‘, 5’-cyclic monophosphate. J.Supramol. Struct. 5:91-102.

Owens, J. R. and Haley, B. E. (1978). Use of nucleo-

tide photoaffinity analogs to determine the

mechanism of ATP regulation of a membrane

bound, cAMP activated protein kinase. 3. Supra-mol. Struct. 9:57-68.

Pariset, C. C., Roussel, C., Weinman, S. 3. and Demail-Ic, J. C. (1983). Calmodulin intracellular concen-

tration and cAMP-dependent protein kinase activ-ity in human sperm samples in relation to sperm

morphology and motility. Gamete Res. 8:171-

182.

Paz (Frenkel), C., Kaplan, R. Vedwab, G., Homannai,

Z. T. and Kraicer, P. F. (1978). Effect of caffeine

on rat epididymal spermatozoa: motility, metab-

olism, and fertilizing capacity. Int. J. Androl.1: 145-15 2.

Perreault, S. D. and Rogers, B. J. (1978). Relationship

between fertilizing ability and cAMP in human

spermatozoa. J. Androl. 3:396-401.

Potter, R. L. and Haley, B. E. (1982). Photoaffinity

labeling of nucleotide binding sites with 8-azido-purine analogs: techniques and applications.

Methods Enzymol. 91:613-633.

Potter, R. L. and Taylor, S. S. (1980). The structuraldomains of cAMP-dependent protein kinase, 1. 3.

Biol. Chem. 255:9700-9712.

Podestra, E. J., Dufau, M. L., Solano, A. R. and Catt,

K. J. (1978). Cyclic adenosine 3’,5’-monophos-phate dependent protein kinase of rat Leydig

cells: physical characteristics of two holoenzymes

and their subunits. J. Biol. Chem. 253:8994-

9001.Rannels, S. R. and Corbin, J. D. (1980). Two different

intrachain cAMP binding sites of cAMP-dependent

protein kinases. 3. Biol. Chem. 255:1083-1088.

Rosado, A., Huacuja, L., Delgado, N. M., Hicks, 3. J.

and Pancardo, R. M. (1976). Cyclic AMP recep-tors in the human spermatozoa membrane. Life

Sci. 17:1707-1714.

Schoff. P. K. (1981). Mammalian sperm cAMP-depen-dent protein kinases: Topological distribution

and control by cAMP and calcium. Ph.D. Thesis.

University of Wyoming. Laramie, WY 82071.

Schoff, P. K., Atherton, R. W. and Haley, B. E.

(1979). A study of the cAMP receptor proteins

of human ejaculated sperm using a photoaffinity

analog. Biol. Reprod. 20:21a.

Schoff, P. K., Forrester, I. T., Haley, B. E. and Ather-ton, R. W. (1982). A study of cAMP binding

proteins on intact and disrupted sperm cells using

8-azidoadenosine 3’,5’-cyclic monophosphate. J.

Cell. Biochem. 19:1-15.Tamblyn, T. M. and First, N. L. (1977). Caffeine-

stimulated ATP-reactivated motility in a deter-

gent-treated bovine sperm model. Arch. Biochem.

Biophys. 181:208-215.

Tash, J. S. and Means, A. R. (1983). Cyclic adenosine

3’, 5’-monophosphate, calcium, and protein phos-phorylation in flagellar motion. Biol. Reprod.

28: 75-105.Wyker, R. and Howards, S. S. (1977). Micropuncture

studies of the motility of mete testis and epididy-mal spermatozoa. Fertil. Steril. 28:108-112.

Zoller, M. 3., Kerlavage, A. R. and Taylor, S. S.

(1979). Structural comparisons of cAMP-depen-

dent protein kinases I and II from porcine

skeletal muscle. 3. Biol. Chem. 254:2408-2412.