Isolation and enzymic properties of the midpiece of bull spermatozoa

Experimental

Cell

Research

38, 217-286

ISOLATION AND MIDPIECE H. The

ENZYMIC OF BULL

MOHRI,2

Wenner-Gren

217

(1965)

Institute,

T.

MOHRI

and

University Received

PROPERTIES OF THE SPERMATOZOA1

July

L.

ERNSTER

of Stockholm,

Stockholm,

Sweden

1, 1964

are among the most highly specialized cells, characterized by their vigorous movement, peculiar structure, and function at fertilization. There is a nice compartmentation in sperm cells: the head contains the hereditary information; the midpiece consists of mitochondria or their derivatives; and the tail is the motile element. During the past thirty years many efforts have been concentrated on clarifying the mechanism of sperm motility, especially by comparison with muscle contraction [7]. One of the basic problems, namely, how the energy necessary for their movement is provided, however, has not yet been completely resolved. It is generally believed that mammalian spermatozoa, supplied with a sufficient amount of glycolyzable sugars in the seminal plasma, obtain their energy for motility preferentially from glycolytic reactions both aerobically and anaerobically [7, 601. This is in contrast to spermatozoa of marine invertebrates, whose motility depends upon the aerobic oxidation of endogenous substrates, mainly phospholipids [64, 66, 801. In the absence of the seminal plasma, hoivever, mammalian spermatozoa can move only in the presence of oxygen, and the substrate for respiration in this case has been shown to be also endogenous phospholipids [35, 46, 47, 49, 531. While there is good evidence that mammalian spermatozoa possess the usual glycolytic pathway [60], less is known about the oxidative stages of spermatozoan metabolism. Among various substrates examined, pyrusate, lactate, members of the Krebs cycle, acetate, propionate, butyrate, acetoacetate, @-hpdroxybutyrate and egg phospholipids have been found to increase the oxygen consumption or to extend motility of bull or ram spermatozoa [44, 49, 511. Experiments using W-labeled substrates indicate the oxidation of acetate, octanoate and butyrate by bull spermatozoa with the production of 14C0,, although only acetate inSPERXIATOZO.I

1 L4bbreuiations: NAD+, oxidized nicotinamide adenine dinucleotide; NADH, its reduced form; NADPf, oxidized nicotinamide adenine dinucleotide phosphate; EDTA, ethylenediaminetctraacetate; Tris, tris (hydroxymethyl) aminomethane. 2 Fellow of the Lalor Founclation (1963-1964). Permanent address: Biological Institute, College of General Education, University of Tokyo, Tokyo, Japan. 15 - 681813

Experimental

Cell Research

38

218

H. Mohri,

T. Mohri

and L. Ernster

creases the oxygen consumption [30]. Concerning the oxidation of pyruvate, the presence of a dismutation reaction, two molecules of pyruvate being converted to acetyl-coenzyme A, lactate and CO,, has been claimed in bull spermatozoa [88]. Furthermore, the oxidation of glycerol, glycerol-l-phosphate and glycerol-2-phosphate, with concomitant accumulation of lactate, has been reported in bull and ram spermatozoa [61, 921. Examination of respiratory pigments revealed the presence of cytochromes a3, a, b, c and c1 both in mammalian and marine invertebrate spermatozoa in the amounts comparable to those found in the cells of other tissues [31, 57, 591. Although little is known about the function of coenzyme Q in sperm cells, it has been reported that coenzyme Q extends motility of fowl spermatozoa [75]. The respiratory chain of spermatozoa responds to amytal and antimycin A with “crossover points” which are typical of these agents [31]. 2,4-Dinitrophenol stimulates respiration and suppresses motility of spermatozoa of several species, probably by uncoupling phosphorylation from respiration [31, 62, 63, 771. It is now firmly established that mitochondria are the site for the Krebs cycle and the oxidative phosphorylation through the respiratory chain in most cells. In mature mammalian spermatozoa, mitochondria are located exclusively in the midpiece region, forming a so-called “mitochondrial sheath” around the base of the tail [29]. According to recent electron microscopical observations, there is a great modification of mitochondrial ultrastructure during spermatogenesis [l]. The question then arises as to whether these specialized mitochondria still exhibit biochemical properties which are found in ordinary mitochondria of other tissues. Up to the present time, biochemical studies of isolated midpieces or mitochondria from spermatozoa are rather scanty. In a pioneer work with sonically disintegrated bull-sperm fragments, Zittle and Zitin [99] have found that cytochrome oxidase activity is present in both the midpiece and the tail fractions, but practically not in the head fraction. Nelson [70] obtained the same distribution pattern with respect to the activities of both cytochrome oxidase and succinic dehydrogenase in bull spermatozoa, and further reported [ 711 that succinic dehydrogenase was concentrated in the nine peripheral fibers of epididymal rat-sperm tail, by examining the frozen-dried section cytochemically with the electron microscope. More recent cytochemica1 studies with light and electron microscopes, however, demonstrated the existence of succinic dehydrogenase in the mitochondrial components of spermatozoa from various invertebrates [l, 341, as well as from rabbit [24] and bull [72]. Experimental

Cell Research

38

Jlitochondrial

functions

219

of bull spermatozoa

In the present experiments the midpieces were isolated from ejaculated bull spermatozoa and their biochemical properties were examined with respect to well-established mitochondrial functions. The results to be described show that the isolated midpieces exhibit oxidation and phosphorylation activities that are similar to those found with isolated mitochondria from animal tissues in general and skeletal muscle in particular. Some implications of these results for the energy metabolism of mammalian spermatozoa \vill be considered. Preliminary accounts of this work have been reported [67, 681.

MATERIAL

AND

METHODS

Preparation of midpiece fraction.PBull semen, diluted with an equal volume of citrate-buffered 20 per cent egg yolk, was obtained from Stockholms Lgns Centrala SeminfGrening, Drottningholm, in the chilled state. The spermatozoa were collected by centrifuging at 2000 xg for 10 min and the supernatant was discarded. The cells were washed free from the remaining seminal plasma with calcium-free “Ringer solution for spermatozoa” [5S] in two more runs at 2000 xg. The packed sperm was weighed, suspended in about 10 volumes of 0.25 M sucrose solution containing 1 mM EDTA, and fractionated by a modification of the method described by Nelson [69] as shown in Table I. Disintegration was made with the aid of a 10 KC Raytheon sonic oscillator (instead of grinding the frozen material in a mortar, in order to avoid any possible damage of mitochondria owing to freezing and thawing). The suspension was saturated with nitrogen prior to the sonication. After removal of the supernatant containing fragmented tails, it was easy to distinguish two layers of the sediment (R2 and R, in Table I): an upper pink layer of midpieces, and a bottom pure-white layer of heads. Usually, a small amount of another white sediment was found on top of these two layers. As microscopical observation indicated that this sediment mainly consisted of large fragments of tails, it was removed carefully by very gentle shaking with a small volume of the sucrose-EDTA medium, and either discarded or combined with the 900 xg supernatant fraction. This combined fraction (T,) was used for the distribution studies of some enzymes and of nitrogen. For electron microscopical examination the tail fragments were collected by a Spinco Model L ultracentrifuge at 100,000 xg for 60 min, leaving the soluble supernatant fraction. The midpiece pellet was separated from the more tightly-packed head fraction by repeated shaking up in sucrose-EDTA medium and recentrifugation. The final pellet of midpieceswas suspended in the sucrose-EDTA medium to give a protein concentration of about 25 mg/ml. All preparative procedures were carried out at 0-4°C. Yields of the head, midpiece and tail fractions obtained with the present method averaged respectively 52, 18 and 30 per cent of the whole spermatozoa by the nitrogen content. These figures are comparable with 51, 16 and 33 per cent obtained by Zittle and O’Dell [98], and 52, 12 and 34 per cent reported by Nelson [70]. Respiration and oxidative phosphorylation.-Oxygen consumption was measured at 30°C by the conventional Warburg technique and, in somecases,polarographically [15]. For the manometric measurements, the standard reaction medium contained Erperimenfal

Cell Research

38

H. Mohri,

220

T. Mohri

und L. Ernster

(unless otherwise stated) 50 pmoles of KCI, 25 pmoles of Tris buffer, pH 7.5, 8 pmoles of MgCl,, 2 mg of bovine serum albumin, 50 pmoles of potassium phosphate buffer, pH 7.5, 2 pmoles of ATP, 30 pmoles of glucose, an excess of yeast hexokinase (Sigma Type III), substrate as indicated, and midpieces (2-3 mg of protein), in a final volume of I ml. In the polarographic experiments, the composition of the medium was as follows: 500 pmoles of sucrose, 150 pmoles of Tris buffer, pII 7.5, 24 TABLE I. Scheme for frnctionntion

of bull spernmtozocr.

Semen Spun 10 min, 2000 xg Washed with Ca-free sperm Ringer, twice Seminal plasma and washings

Sperm Suspended in sucroseEDTA, 2 min sonication, Spun 10 min, 9ooxg

I RI

d1 Suspended in sucroseEDTA, Spun 10 min, 9ooxg !

R2 Gently amount

shaken with small of sucrose-EDTA

Combined T,

I HI

Ml Suspended in sucroseEDTA, Spun 10 min, 9ooxg

Spun 60 min, 100,000 x g I T2

S5

Tuil

I R,

Supernatant

S, Separated

as above

HZ

M2 I

Head

Combined Spun 10 min, I %

Mid-piece Experimental

Y--

Cell

Research

38

S4 Discarded

900 x g

Jlitochondrial

functions

of bull spermatozorc

221

pmoles of MgCl,, 2 pmoles of EDTA, 5 pmoles of potassium phosphate buffer, pH 7.5, substrate as indicated, and midpieces (3-5 mg of protein) in a final volume of 3 ml. As the phosphate acceptor, 5 pmoles of ADP were added. The respiration rate, of protein. The term Zol designates Q is expressed as pl of 0, consumed/hr/mg t,“: number of pl of 0, consumed/hr/lOs spermatozoa (cf. 1761). Phosphorylation was determined by the isotope distribution method [54]. In calculation of P: 0 ratios, the amount of oxygen consumed (as determined manometrically) was corrected by direct extrapolation for 5 min of thermoequilibration. The duration of incubation was 30 or 60 min. FrucloZysis.-Fructolysis was followed by incubating anaerobically the whole spermatozoa in a medium recommended by Rothschild [79] for 90 min at 37°C. The gas phase was a mixture of 95 per cent nitrogen and 5 per cent carbon dioxide. The products were analyzed after deproteinization with 6 per cent perchloric acid and removal of perchlorate after the addition of K&OS. Chemical and enzymatic determinations.-The nitrogen content was determined by direct Nesslerization after the combustion of samples with H,SO, and H,O,. The amount of protein was obtained by multiplying the nitrogen content by 6.25. Fructose was measured according to the method of Rothschild [ 781. Glycerol was estimated by the method of Ryley [81]. Lactate [39], pyruvate [12], dihydroxyacetone phosphate [IO] and glycerol-l-phosphate [38] were determined enzymatically by following the changes at 340 rnp, owing to the reduction of NAD-1. or the oxidation of NADH, in a Beckman DK-2 recording spectrophotometer. Enzyme assays.-NADH oxidase activity of the midpiece fraction was followed by measuring the decrease in absorbancy at 340 rnp in a system containing 375 pmoles of sucrose, 150 pmoles of Tris buffer, pH 7.5, 24 pmoles of MgCl,, 5 pmoles of potassium phosphate buffer, pH 7.5, 5 pmoles of ADP, 0.3 pmoles of NADH and the midpieces in a final volume of 3 ml, or manometrically as described above using 5 ,umoles of NADH. Activities of hexokinase [3], malic dehydrogenase 1741, lactic dehydrogenase and glycerol-l-phosphate dehydrogenase [S], and aldolase and glyceraldehyde-3-phosphate dehydrogenase [95] were all assayed spectrophotometrically by measuring the absorbancy change at 340 rnp, due to the oxidation or the reduction of pyridine nucleotides. Glycerokinase activity was assayed by the method of Bublitz and Kennedy [9]. Chemicals.-All chemicals and enzymes were commercially available products. Deionized water was used to prepare all solutions. Electron micrographs.-For electron microscopical examinations, the packed sperm and the pellets of fragments after centrifugation were fixed by immersion for one hour in Kellenberger’s [42] fixative for bacteria and other microorganisms, containing 1 per cent osmium tetroxide. After dehydration in a graded series of increasing concentrations of ethanol, the samples were embedded in Epon. Thin sections were made with an LKB “Ultrotome” ultramicrotome at a thickness of 48 to 60 p. Electron micrographs were taken with a Hitachi electron microscope, Model HS-6, at original magnifications ranging from x 4000 to x 7000. Sperm count.-The number of spermatozoa was counted by means of a Thoma ruling hemocytometer.

Experimental

Cell

Research

38

222

H. Mohri,

T. Mohri

and

I,. Ernster

RESULTS dlorphologicrrl obsermtiom-Electron microscopical survey of each fraction (Figs. 1-4) showed a purity of more than 90 per cent. In the midpiece fraction, contaminants were a very few number of heads and fragments of tails, especially the part of principal piece adjacent to the midpiece. .Is can be seen from comparison with the electron micrograph of the whole spermatozoa, the isolated midpieces appeared to be morphologically intact, except that most of them lost the outer cell membrane. Wren after the sonic treatment. which is usually employed to disrupt ordinary mitochondria, no visible change took place in the mitochondrial sheath of spermatozoa. The midpiece fraction n-as not contaminated with any sub-mitochondrial fragments. The tails, on the other hand, were fragmented into very fine pieces. The typical 9 + 2 structure \vas lost, and empty fibrous sheaths of very short length were found abundantly. This fraction seems to contain some free mitochondria and fragments of cell membrane. Contaminants were only rarely found in the head fraction. Strrndrrrdixtion of’midpiece frnction-As preliminary experiments revealed that the biuret reaction and other methods for protein determination \vere not applicable to the sperm midpieces because of their resistance to alkali and other treatments, the turbidity of the suspension was measured at 520 m,u to standardize the concentration of midpiece to be used. I’sually, 25 ,~l of the final dense suspension of midpieces \yas diluted to ci ml with the sucroscEDTA medium for the measurements. Linear relationships \verc obtained between the amount of midpiece and ()I),,,, (Fig. 5 rr), and between the nitrogen content measured by the Nessler procedure and OD,,,, (Fig. 5h). Respiration rrntl oxidative phosphorylrrtior~.~‘I’he Z,,? of washed whole spermatozoa measured polarographically at 30°C in the calcium-free “Hingerphosphate” solution [32] \vas ‘L-3. .L\ Xoi value of about 1 has been reported by Gonse [31] under similar experimental conditions at 23°C. A\s shown in Fig. 6, the oxygen consumption inc,reased in proportion to the amount of midpiece fraction added. The oxidation rate \vas almost constant during the incubation period up to 60 min, provided that the amount of substrate was sufficient. In Table II are summarized the oxidation rates, Qo1, and the P: 0 ratios

Fig.

l.-Electron

micrograph

of the washed

bull

Fig.

2.-Electron

micrograph

of the isolated

head

Experimental

Cell Research

38

spermatozoa. fraction.

Y 15,000. x 15,000.

Mitochondrial

functions

of’ bull spermcrtoroa

223

224

H. Jlohri,

T. Mohri

and I>. Ernster

obtained with several substrates, using the midpiece fraction. All data listed in the table were calculated from manometric determinations. It is readily noticeable that, among the substrates tested, glycerol-l-phosphate \\-as oxidized by the sperm midpieces at the highest rates both in the presence and the absence of cytochrome c (fnal cont., 30 ELM). Other substrates, such as various Krebs cycle intermediates, glutamate and P-hydroxybutyrate, were oxidized at appreciable but slower rates. Pyruvate was oxidized only slo\vly if it was added alone, but its oxidation was much accelerated by adding malate. The rate with pyruvate plus malate varied rather greatly among semen samples. This seems to be due to seasonal variations, since the high rates with these substrates were obtained only during a certain restricted period, in January and February, in the course of the present studies. It has been reported that there is a great variation in the ability of difIercnt samples of intact bull spermatozoa to oxidize pyruvate [62]. These variations have been correlated to the fertilizing power of semen, while in the present experiments semen samples only from bulls showing good fertility were used. For the oxidation of citrate and isocitrate, the addition of either SAD’ or NADPf was required, both ppridine nucleotides stimulating the oxidation to about the same extent. NAD+ and NADP+, however, had no effect on the oxidation of pyruvate plus malate. The P: 0 ratios obtained with these substrates were close to the generally accepted values, i.e., 2 with glycerol-l-phosphate or succinate, and 3 with pyridine nucleotide-linked substrates. Although these values vvere observed in the presence of serum albumin, which is known to bind endogenous uncoupling agents [94], the addition of serum albumin was not absolutely required in demonstrating efficient coupled phosphorylation. For example, the P: 0 ratios with glycerol-l-phosphate, succinate, and pyruvate plus malate in the absence of cptochrome c were 1.42, 1.58 and 2.31, respectively, in the absence of albumin, and 1.57, 1.57 and 2.42, respectively, in its presence. Addition of cytochrome c remarkably enhanced the oxidation of glycerol- lphosphate and succinate by the midpiece fraction, while this was not the case with other substrates. Consequently, the oxidation rate with succinate far exceeded that with pyruvate plus malate, which gave the highest respiration among the Krebs cycle intermediates in the absence of added cytochrome c. The results of a typical experiment obtained by polarographic measure-

Fig.

3.-Electron

micrograph

of the isolated

midpiece

Fig.

4.-Electron

micrograph

of the isolated

tail

Experimental

Cell Research

38

fraction.

fraction.

x 15,000.

x 15,000.

Mitochondrinl

functions

of bull spermatozoa

Experimental

223

Cell

Research

38

226

H. Mohri,

T. Mohri

and II. Ernster

ment are shown in Fig. 7. The maximum stimulation by cytochrome c was obtained at a final concentration of 10 /AM both with glycerol-l-phosphate and succinate. In this experiment, the oxidation of pyruvate plus malate was not improved at any concentration of cytochrome c examined. A similar Ok,

a

b

0.50

0.25

/

I

0

I

0.05

0.1

0

I

,

50 W TOTAL N

MI.

I

100

Fig. 5.-Standardization of the isolated midpiece fraction. (a) Relationship between the amount of midpieces and the optical density at 520 rnp. (b) Relationship between the total nitrogen content and the optical density at 520 rn,&

G-l-P

50

25 Pyr. + Mal. * I

0

I

I

I

I

0.1

0.2

0

CCyt.cl

Ml.

Fig. Fig. B.-Relationship Substrate, pyruvate

6.

between (20 mM)

Fig. 7.-Effect of cytochrome (20 m&f) and pyruvate (20 mM) Experimental

Cell Research

10‘5

10‘6

38

Fig.

the amount plus malate

of bull-sperm (20 mM).

midpieces

7.

and the oxygen

consumption.

c on the oxidation of glycerol-l-phosphate (50 mm), succinate plus malate (20 mA4). Concentration of cytochrome c given in 31.

Mitochondrial

functions

of bull spermatozoa

227

curve was obtained with glutamate. In manometric experiments, ho\yever, a slight increase after the addition of cytochrome c was observed in the oxidation of the latter two substrates (Table II). Such a stimulation of oxygen consumption by cytochrome c does not appear to be due to contamination by sub-mitochondrial fragments in the midpiece fraction, judging from the electron micrographs. 4s can be seen from Table II, the stimulation by cytochrome c of the oxidation of both glycerol-l -phosphate and succinate was accompanied by a decrease in the I’: 0 ratios. =\gain, this was not the case with pyridine nucleotide-linked substrates. The enhanced part of oxygen ‘TARLE II.

Oxidative

phosphorylation No. of exp.

(~1 O,/hr/mg

3

1.0

None QT.

c (30

,/AI)

Succiuatc

(20

-; qt. I’yruvate

(20 + cyt. -I- NAD+

Glutamate -i qt. Pyruvate

(20

Malate

(20

Citrate

Isocitr,ltc

(1.2-1.7)

6

15.4

(12.2-19.0)

I.7

(1.4-1.9)

/AM)

1

55.0

(48.5-63.1)

0.9

(0.8-0.9)

10

B-H\-droxybutyrate

m.11)

/AlI)

m.ZI)

1nJf) mu)

m-11)

(1.5 (20

-’ ShDP+ u-Ketoglutaratc

+ malate m.11)

(I.5

-. NADf

(0.4-2.7)

(0.9-1.1)

mJZ)

+KADP+

1.2

1.0

(1.5

(20

4.3)

(55.1-84.8)

(1.5

-tkKAD+

(l.l-

69.9

m.71)

~ SXDPf

(0.6-2.8)

8

mJI)

--SAD’

1.8

/Au)

p.lf)

c (30

1.8)

1.5

(l..i

(20

(0.5-

(27.0-49.6)

(I.5

--- NAL>P+

P:O

2.7

m.11)

c (30

protein)

36.4

mJ1)

mJ1)

c (30

(20%

3 (50

c (30

midpieces.

10

Glycerol-l-phosphate -q-t.

by isolated bull-sperm

m;ll)

m.\I) (I.5

(20

mN)

21.2

(9.4-44.6)

2.5

(2.2-3.2)

6

32.8

(15.7-45.6)

2.4

(2.2-2.6)

2

26.0

(8.9-43.1)

2.4

(2.3-2.5)

‘2

27.7

(9.2-46.2)

2.1

(2.1-2.1)

5

15.7

(10.3-18.7)

2.6

(2.1-3.2)

3

18.4

(16.6-20.0)

2.3

(2.1-2.4)

‘2

4.7

(4.3-

2.1

(1.9-2.3)

1

9.2

(6.8-12.7)

2.5

(2.2-2.6)

1

9.9

2.1

1

8.0

2.1

5.1)

6

2.9

(I&

4.0)

2.3

(1.1-3.2)

2

5.1

(4.1-

6.1)

1.X

(1.1-2.4)

3

X.0

(5.7-19.0)

1.3

(1.1-1.4)

9

3.6

(2.3-

2.3

(1.9-3.0)

4

9.2

(6.9-12.0)

2.0

(1.8-2.2)

m.-ll)

5

10.9

(X.0-17.2)

1.9

(1.9-2.0)

m.lf)

::

13.8

2.5

(2.0-3.0)

I.5

(0.5-1.9)

mM)

(1.5

(20

(20

mJ1)

4

6.6

5.i)

(11.3-15.1) (2.9-17.6)

Experimentd

Cell

Keseurch

38

H. Mohri,

228

T. Mohri

und L. Ernster

consumption with glycerol-l -phosphate or succinate as substrates, however, was not completely devoid of coupled phosphorylation. The addition of cytochrome c always caused a definite increase in the absolute amount of phosphate esterified, e.g., the amounts of phosphate esterifiedxvith and without cytochrome c were 8.76 and 5.90 /Amoles, respectively, for glycerol-l -phosphate, and 5.65 and 4.01 ymoles for succinate. In connection with the stimulating action of cytochrome c, it should be mentioned that cgtochrome c easily leaks out from the sperm structure as a result of cellular damage or prolonged storage of spermatozoa [59]. Since a preliminary spectrophotometric examination indicated the existence of cytochromes U, b and c in the isolated midpiece fraction, the midpieces or mitochondria seem to be only partially deficient in cytochrome c. The same examination revealed the presence of cytochrome c in the tail (‘I’,) fraction. It is still obscure, however, whether this n-as detached from the mitochondria during preparation or pre-existed in the tails. As already mentioned in the introduction and can also be seen from the electron micrographs (Figs. 1 and 3), the midpiece of bull spermatozoa con“mitochondrial sheath” surrounding the beginning part of tail. sists of a If we assume that the tail has no or only feeble respiratory activity, and that the external and internal diameters of the “mitochondrial sheath” are 0.9 and 0.5 ,u, respectively [31], the oxidation rates listed in Table II could be multiplied by about one and a half on the basis of protein of mitochondria. One experiment \vas carried out at two different temperatures, viz. 30°C and 37°C. The Qo, values obtained with glycerol-l -phosphate, succinate and pyruvate plus malate in the presence of cytochrome L’ were as follows: 72.4, 63.1 and 45.6 at 3O”C, and 119.6, 109.8 and 70.5 at 37”C, respectively. From these data an average Q,, between 30°C and 37°C can be calculated as 2.35. Since the midpiece constitutes 18 per cent of the whole spermatozoon by the nitrogen content, and 108 spermatozoa were found to correspond to 1.46 mg of protein, the latter three figures above correspond to .Z,% values of 31.4, 28.8 and 18.5. These values fall into the upper range of endogenous respiration of Lvhole bull spermatozoa so far measured at 37”C, 3-30 [32, 48, 621. In other words, under optimal conditions the isolated midpieces oxidize the above substrates at rates sufficient to account for the respiration of intact spermatozoa. Respiratory control.-Since the first demonstration by Lard? and \Vellman [SO] it has been widely recognized that both inorganic phosphate and the phosphate acceptor, ADP, are required for maximal respiration of isolated mitochondria from various tissues. This is also the case with the isolated Experimental

Cell

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38

Mitochondrial

functions

of bull spermatozorr

229

midpiece fraction as shown in Figs. 8 and 9. Four different substrates were tested, and ATP + hexokinase + glucose was used as the terminal phosphate acceptor. In a separate experiment, concentrations of inorganic phosphate ranging from 5 to 50 mM gave the same oxidation rate with pyruvate plus malate as substrate. It can be noted that in the cases of glycerol-l-phosphate and succinate the respiratory control almost disappeared upon the addition of cytochrome c, in accordance with the decrease in P : 0 ratios. The respiratory control indexes ( =the ratios of 0, consumed in the presence of phosphate acceptor to 0, consumed in the absence of phosphate acceptor) calculated from the data in Fig. 8 \vere 1.04 and 3.39 for glycerol-l-phosphate, and 1.13 and 1.87 for succinate, in the presence and absence of cytochrome c respectively. With pyridine nucleotide-linked substrates, on the other hand, rather good respiratory control was still observable in the presence of cyto-

15

+c;t.c

Pj’r. + Mal. -wt. c

10

6

0” i

/&

i

20

10

5

10

15

30

15

Time (mini I:ig.

8.

30

15

30 Time [min)

Fig.

15

30

9.

Fig. 8.-Respiratory control in bull-sperm midpieces in the presence and the absence of added cytochrome c (30 pM). Substrates, glycerol-l-phosphate (50 m&Z) and succinate (20 mhl). o, the complete system; A, minus phosphate acceptor; x , minus inorganic phosphate; 0, minus inorganic phosphate and phosphate acceptor. Fig. S.-Respiratory control in bull-sperm cytochrome e (30$W). Substrates, pyruvate Symbols are the same as in Fig. 8.

midpieces in the presence and the absence (20 mM) plus malate (20 mM) and glutamate

Experimenfnl

of added (20 m&f).

Cell Research

38

230

H. Mohri,

chrome c, consistent with ved in the presence and control indexes calculated plus malate, and 11.54 absence of cytochrome c TABLE

III.

T. Mohri

and L. Ernster

the insignificant differences in the I? : 0 ratios obserabsence of added cytochrome c. The respiratory from Fig. 9 were 3.48 and 7.72 with pyruvate and 10.07 with glutamate, in the presence and respectively.

Effects of rotenone, amytal and antimycin A on the oxidation glycerol-l-phosphate, succinate and pyruvate plus malate by the isolated bull-sperm midpieces. Qo,

(,ul O,/hr/mg + Cyt.

c

Inhibition (%)

protein) - Cyt.

c

+ cyt.

c

- Cyt.

Glycerol-l-phosphate (50 mM) + rotenone (1yM) +amytal (2 mM) +antimycin A (3 ,uM)

65.0 61.2 58.8 6.4

40.1 41.3 38.8 2.9

5.7 9.9 90.2

3.7 92.7

Succinate (20 mM) + rotenone + amytal + antimycin A

54.9 48.2 39.5 2.6

15.9 16.9 14.1 3.2

12.5 28.2 95.4

(7.2) 12.3 79.8

34.3 3.3 2.5 4.0

27.9 1.3 2.5 1.0

90.4 92.7 88.2

95.3

Pyruvate (20 mM) + malate + rotenone + amytal + antimycin A

(20 mM)

of

c

(2.9)

91.0 96.5

Effect of inhibitors.-In order to obtain more precise information about the respiratory chain of the sperm mitochondria, the effects of several inhibitors mere examined with the midpiece fraction. Among them, amytal [13, 271 and rotenone [26, 53, 731 have been known as specific inhibitors of electron transfer between NAD and flavoprotein, while antimycin -4 acts on the step between cytochromes b and c [16]. Table III clearly shows that the oxidation of both glycerol-l-phosphate and succinate was almost completely inhibited by antimycin A, but was affected only slightly by rotenone and amytal, whereas the oxidation of pyruvate plus malate, which are NAD-linked substrates, was nearly completely inhibited by all three agents. No significant difference vvas observed between the data obtained with and without cytochrome c, except that in the presence of cytochrome c the inhibitory action Experimental

Cell Research

38

Mitochondrial

functions

of bull spermatozoa

231

of rotenone and amytal on the oxidation of glycerol-l-phosphate or succinate was somewhat more pronounced, and that in the absence of cytochrome c rotenone caused a slight stimulation instead of inhibition of the oxidation of these two substrates. The results are thus consistent with the above mentioned actions of these inhibitors. Gonse [31] has reported inhibition of respiration of TABLE

IV. Effects of 2,4-dinitrophenol (DNP) and oligomycin on respiration and phosphorylation by the isolated bull-sperm midpieces.

(,A O,/hr/Zi

Glycerol-l-phosphate (50 mM) + DNP (10 @f) +DNP (100 ,uM) i DNP (1 m&I) Pyruvate (20 mM)+malate + DNP (10 ,uM) i DNP (100 ,uM) i DNP (1 mM) Glycerol-l-phosphate + DNP (100 -k oligomycin

(20 mM)

(50 mM) /A4) (1 pg/mg of protein)

Pyruvate (20 mM)+malate + DNP (100 ,u”M) +oligomycin (1 pg/mg

(20 mM) of protein)

protein)

P :0

22.1 22.7 23.1 21.9

1.30 1.16 0.49 0.03

9.2 9.7 6.8 2.0

2.15 1.80 0.65 0.20

32.6 30.0 9.5

1.60 0.70 0.37

14.8 10.7 5.5

2.09 0.54 0.51

bull spermatozoa by antimycin A in the presence of lactate as substrate, and no inhibition of respiration by amytal in the presence of succinate. The effects of 2,4-dinitrophenol and oligomycin on the oxidation rate and the P: 0 ratio are summarized in Table IV. In contrast to the case with glycerol-l-phosphate as substrate, where 2,4-dinitrophenol caused an almost complete uncoupling without affecting the oxygen consumption, the oxidation of pyruvate plus malate was diminished by increasing the concentration of 2,4-dinitrophenol, together with a lowering in the P: 0 ratio. 4t a concentration of 10 ,&f, however, 2,4-dinitrophenol induced a maximal level of oxygen consumption with both substrates and uncoupled phosphorylation to a slight extent. Similar observations have been reported with mitochondria from skeletal muscle [a]. In washed bull spermatozoa, the optimum concentration of 2,4-dinitrophenol inducing maximal activation of respiration has been Experimental

Cell

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38

H. Mohri,

232

T. Mohri and I,. Ernster

reported to be 100 to 200 ,LL%‘,when lactate was added as substrate [31]. Oligomycin at a concentration of 1 /bg/rng of protein inhibited tightly-coupled respiration and abolished phosphorylation by the midpiece fraction, in accordance with its known effect [40, 453. Oxidation of glgcerol-l-phosphate.PAs described in a foregoing section, the bull sperm midpieces oxidize glycerol-l-phosphate at the highest rate. This is also a peculiar feature of insect flight-muscle mitochondria, and glycerol-l-phosphate has been considered as the most important source of energy for insect flight ‘14, 82, 831, although evidence is accumulating that the Krebs cycle intermediates as well may be important sources of energy [33, 911. In connection with the oxidation of glycerol-l-phosphate in insect sarcosomes, it has been shown that the rate of oxidation tends to decrease with time and becomes very low \vhen the ratio (glycerol-l-phosphate/ dihydroxyacetone phosphate) reaches near 3 [6, 19]. The results of an examination of this possibility in the sperm midpieces are presented in Fig. 10, where dotted lines indicate the theoretical amount of oxygen consumption calculated for the complete conversion of r.-glycerol-l-phosphate into dihydroxyacetone phosphate and corrected for the cndogenous respiration. Quantitative enzymatic analysis [38] showed that the sample of nL-glycerol-lphosphate (Sigma Chemical Co.) used in this experiment contained 47.0 per cent of the biochemically active I.-isomer. It is evident that L-glycerol-lphosphate was oxidized stoichiometrically to dihgdroxyacetone phosphate by the midpiece fraction. The addition of more glycerol-l-phosphate after a plateau was obtained evoked a second burst of oxygen uptake, followed by a new plateau. A commercially available sample of glycerol-2-phosphate was oxidized by the midpieces at exactly the same rate as glycerol-l-phosphate. In this case, ho\vever, a plateau was attained at much lower level than in the case of glycerol-l-phosphate. Enzymatic analysis revealed that this product contained the corresponding amount of I.-glycerol-l-phosphate. Free glycerol \vas not oxidized by the sperm midpieces at any appreciable rate. It may be mentioned here that the respiration of washed whole spermatozoa \vas also increased by adding glycerol-l-phosphate (cf. also [61]) and further stimulated by cytochrome c. As summarized in Table V, a further enhancement of the oxidation rate was obtained by combining glycerol-l-phosphate and succinate. The rate with these two substrates was approximately equal to the sum of the rates obtained with the substrates when tested individually in the absence of added cytochrome c. Even in the presence of cytochrome c, some enhancement was observed by the combination of the two substrates. The results seem Experimental

Cell Research

38

Mitochondrial

functions

of bull spermatozoa

233

to suggest that the terminal oxidation of glycerol-l-phosphate and of succinate proceeds at least partly by different respiratory chains. Oxidation of isocitrate.-It has been stated above that the oxidation of both citrate and isocitrate by the sperm midpieces proceeded at a very slo\v rate in the absence of added pyridine nucleotides, either NAD+ or NADPf. The results of another series of experiments are shown in Table VI. It is apparent that both NAD+ and NADP+ are able to enhance the oxidation of isocitrate to the same extent. Combination of NAD+ and N,4DP+ increased the oxidation TABLE

V. Oxidation rates with glycerol-l-phosphate, succinate glycerol-l-phosphate plus succinate in isolated bull-sperm midpieces.

and

Qo, (~1 O,/hr/mg - c&t. Glycerol-l-phosphate Succinate (20 mM) Glycerol-l-phosphate succinate (20 mM)

TABLE

VI.

(50 m&f) (50 n&Z)

+ Cyt.

28.8 10.6

71.1 57.3

36.5

87.3

Effect of hrAD+ and NADP+ on the oxidation of isocitrate, malate and isocitrate plus malate by the isolated bull-sperm midpieces. Qo,

protein)

Tsocilratc (20 m&1) +NAD+ (1.5 mM) + NADP+ (1.5 mM) + NAD+ + NADP+

5.7 12.0 11.2 13.9

2.5 10.2 10.1 11.2

Malate

6.8 10.6 6.9 10.6

7.9 9.9 8.0 11.3

36.8 37.8 39.3 99.0

28.4 26.4 25.4 25.0

(20 mM) -t NAD+ im NADPf + NAD+ + NADPf

Isocitrate (20 m&f) + malate + NAD+ + NADP+ + NAD+ + NADPf

651813

c

+

(/&I O,/hr/mg

16-

protein)

c

(20 m&f)

Experimental

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38

234

H. Mohri.

T. Mohri

and I,. Ernster

rate somewhat further. The oxidation of malate, on the other hand, was enhanced only slightly bp SAD+, and not at all by NADP+. A very remarkable stimulation of the oxygen uptake \vas observed when isocitrate and malate were added in a combination, far exceeding the sum of the oxygen uptake with isocitrate alone and with malate alone. The oxidation rate was comparable to that of pyruvate plus malate (see Table II), and was not enhanced further by the addition of NAD +, SADP+ or ?JAD+ plus NADP+. Sl.ch a stimulation of aerobic oxidation of isocitrate by malate has been reported in rat-liver mitochondria [ 17, 181, and led to the hypothesis that the pyridine nucleotides reduced by the isocitrate dehpdrogenase are not available to the respiratory chain, but must react with oxaloacetate by the action of malate dehydrogenase; the malate is then oxidized via another molecule of pyridine nucleotide which is available to the cytochrome system. If this hypothesis is correct it would be expected that malate is only required in catalytic concentrations to stimulate isocitrate oxidation, but conversely, that catalytic concentrations of isocitrate do not stimulate malate oxidation; furthermore, it would be expected that the effect of malate on isocitrate oxidation is duplicated by oxaloacetate. Data summarized in Table \‘I1 show that this indeed is the case. Both malate and oxaloacetate induced half-maximal rates of isoTABLE VII.

Effect

of malate and oxaloacetate on the oxidation by the isolated bull-sperm midpieces. Qo, (~1 Whr/mg protein)

Isocitrate Malate

(20 m&f) (10 milrl)

Oxaloacetate Isocitrate

Cell Research

(1 mM)

38

3.8

(10 mM) (20 mM)

Isocitrate

Experimenlal

3.0

-t malate (1 +malate (2 i- malate (5 Imalate (10 i- oxaloacelate -t oxaloacetatc I oxaloacetate + oxaloacetate

3.6 mM) m&f) mM) mM) (1 (2 (5 (IO

mM) mM) mM) mM)

+ malate (10 m&f) + oxaloacetalc (10 mM)

10.9 15.1 21.1 22.3 12.9 16.3 15.9 16.1 5.6 7.2

of isocitrate

Mitochondrial

functions

of bull

235

spermnto:on

citrate oxidation in concentrations of about 1 mill, whereas 1 mM isocitrate enhanced the rates of oxygen uptake with malate and oxaloacetate only slightly. Yet, the concentration of malate required for half-maximal rate of isocitrate oxidation was about 10 times higher than that reported by Chappell T4111,~

1’111. NADH

oxidation

by the isolated

No. of cxp.

(~1 O,/hr/mg

midpieces.

vo,

NADH

(5 m&Z) +rotenone (1 ,uM) +antimycin A (3 pM)

3 3 3

3.4 2.0 2.5

NADH

(5 mM) -t rotenone

+ cyl.

3 3

+ antimycin

A

3

c (30 ~ciV1)

bull-sperm

protein)

(1.3(0.6(1.6-

P:O

5.2) 3.5) 3.1)

0.7

(0.2-1.6)

53.4 13.7

(43.6-63.1) (9.2-l 7.5)

0.1

(0.1-0.1) -

14.3

(10.7-16.4)

[1X]. Results similar to those shown in Table ITI1 were obtained also vvith citrate instead of isocitrate. Oxidation of iVADH.-Intact mitochondria from several sources are virtually unable to oxidize externally added NADH [8, 22, 37, 521. As shown in Table 17111,this was also true for the bull-sperm midpieces. The addition of cytochrome c, however, brought about more than 1%fold stimulation of the oxidation of added NADH. The oxidation rate thus obtained was almost the same as those observed with glycerol-l-phosphate and succinate, and exceeded the rate with pyruvate plus malate. This cytochrome c-induced oxidation of NADH was inhibited by both rotenone and antimycin A by more than 70 per cent. Such a rotenone- (amytal-) and antimycin A-senGtive pathway for the oxidation of external NADH, involving extra-mitochondrial cytochrome c, has been so far reported only in mitochondria from rat skeletal muscle [37], whereas in mitochondria from rat liver [25, 26, 521 and human skeletal muscle [37] the cytochrome c-stimulated NAIIH oxidation is virtually insensitive to these agents. In contrast to the latter cases, however, no significant increase in the amount of phosphate esteritied could be demonstrated after the addition of cytochrome c in the present material, the pathway thus appearing to be non-phosphorylating. The same results were obtained by measuring the changes at 340 m,u. In rat liver [41] and skeletal muscle [37] mitochondria, cytochrome ( can be replaced by the lower homologues of coenzyme (2, coenzymes Q. and Experimental

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236

H. Mohri,

T. Mohri

and L. Ernster

QV in

inducing the stimulation of NADH oxidation, whereas the higher homologues gave negative results. As shown in Table IX, similar results were obtained. Coenzyme Q. stimulated the oxidation rate of NADH up to the rate obtained with cytochrome c, while coenzyme Q,, exerted no stimulation. TABLE

IX.

NADH

oxidation

by the isolated bull-sperm

midpieces.

Q% (~1 Whrlmg

NADH

(5 +cyt. +co +Co +Co +Co

mM) c (30 ,uM) 00 (100 PM) Qo (330 ,uM) Q. (1 mM) Qm (330 PM)

protein)

P:O

1.8 34.7 5.2 18.2 34.8 1.0

0.8 0.1 0.4 0.2 0.1 0.9

The concentration of coenzyme Q,, giving rise to the same oxidation rate as that obtained with cytochrome c was rather high. Again, the enhanced respiration was virtually devoid of phosphorylation. Although the presence of cytochrome c has been detected in the tail fraction, it is not yet settled whether bull spermatozoa really contain extramitochondrial cytochrome c or coenzyme Q. The extent to which the above pathways contribute to the oxidation of extra-mitochondrial NADH in intact spermatozoa is therefore not clear. Glycerol-l-phosphate cycle.-Since the sperm midpieces exhibit a high ability to oxidize glycerol-l-phosphate, the possibility of the operation of a “glycerol-l-phosphate cycle” in bull spermatozoa was investigated. This cycle, which involves a cyclic oxidation and reduction of glycerol-l-phosphate by means of the mitochondrial glycerol-l-phosphate oxidase and the soluble NAD-linked glycerol- 1 -phosphate dehydrogenase, resulting in an indirect oxidation of extra-mitochondrial NADH, has originally been postulated to function in insect flight muscle [ 11, 281. More recently, the “glyceroll-phosphate cycle” has been reconstructed with isolated mitochondria and cell-sap from rat skeletal muscle [87]. As can be seen from Table X and Fig. 11, it was easy to reconstruct the “glycerol-l -phosphate cycle” by incubating glycerol- 1 -phosphate and a puritied preparation of rabbit muscle glycerol-l-phosphate dehydrogenase (Raranowski enzyme) together with the midpiece fraction. The data shown in Experimental

Cell

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38

Mifochondrial

functions

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237

Table X were obtained spectrophotometrically by following the decrease in absorbancy at 340 mp, and indicate that the oxidation of NADH can be abolished by antimycin A, but not by either rotenone or amytal. The addition of cytochrome c further stimulated the oxidation of NADH and this was again “Glycerol-l-phosphate cycle” by means of crystalline NAP-linked glycerol-l-phosphate dehydrogenase from rabbit muscle (Baranowski enzyme) and bull-sperm midpieces. TABLE

X.

NADH oxidized (mymoles/ min/mg protein) None Glycerol-l-phosphate Baranowski enzyme Glycerol-l-phosphate Glycerol-l-phosphate Glycerol-l-phosphate Glycerol-1-phosphale Note. antimycin

Final concentrations A, 1 ,ug/ml.

(20 mM) (100 pg) + Baranowski + Baranowski + Baranowski + Baranowski of inhibitors

enzyme enzyme enzyme enzyme were:

+ rotenone t amytal + antimycin rotenone,

1.6 1.9 2.3 21.8 23.0 22.4 3.5

A 1.7 ,uM;

amytal,

2 mM;

only antimycin A-sensitive. Fig. 11, on the other hand, contains curves obtained manometrically and show that, if glycerol-l-phosphate, NADH, and Baranowski enzyme were added in combination, the respiration proceeded at a faster rate than those with glycerol-l-phosphate alone, with glycerol-l -phosphate plus Baranowski enzyme or with glycerol-l -phosphate plus NADH, and far exceeded the theoretical amount of oxygen consumption for the conversion of glycerol-l -phosphate added into dihydroxyacetone phosphate, owing to the operation of “glycerol-l-phosphate cycle”. The most probable source of NAD-linked glycerol-l-phosphate dehydrogenase in bull spermatozoa is the tail fraction, because it has been shown that sperm heads are enzymatically inert [65, 69, 70, 991. The effect of the tail fraction on the oxidation of NADH by the midpiece fraction was therefore investigated. As can be seen from Fig. 11, however, the tail fraction failed to replace the purified NAD-linked glycerol-l-phosphate dehydrogenase, i.e., the oxygen consumption with glycerol-l-phosphate, NADH, and the tail fraction did not exceed that with glycerol-l-phosphate plus NADH. An Experimental

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38

H. Mohri,

208

T. Mohri

crnd I>. Ernster

attempt was also made to demonstrate the activity of Sill>-linked glycerol-lphosphate dehydrogenase in the tail fraction, using dihydroxyacetone phosphate as substrate. It is evident from the data in Table Xl that the tail fraction has little if any activity of this enzyme as compared with the potent activity of lactic dehydrogenase. Seither the midpiece nor the head fraction showed

I

0

20

40 Time (mini

Fig.

‘I

”

1

60

I

x I

80 TimeCminl

10.

Fig. lO.-Oxidation of glycerol-l-DL-phosphate 10 ,umoles; x , 0 ,umolc. Cytochrome c (30 $44) 10 ,umoles of DL-glycerol-l-phosphate.

Fig.

by bull-sperm was added. Arrow

3 1.

midpieces. S, 20 ,~moles; e, indicates the addition of another

Fig. il.-“Glycerol-l-phosphate cycle” by means of crystalline NXD+-linked dehydrogenase from rabbit muscle (Baranowski enzyme) and bull-sperm of tails when added was 2 mg of protein.

glycerol-l-phosphate midpieces. The amount

any activity of the former enzyme. These results suggest that the “glycerol-lphosphate cycle” may not operate in bull spermatozoa. A similar negative result has been reported with Ehrlich ascitei tumor cells [8]. Another attempt to stimulate the oxidation of NADH, by cata!ytic amounts of acetoacetate or @-hydroxybutyrate (“P-hydroxybutyrate cycle” [22’), has so far been unsuccessful. Glycolytic enzymes.-The activities of some key enzymes in glycolysis were found to be concentrated in the tail fraction (Table XII). Lactic dehydrogenase was found not only in the supernatant fraction obtained after the centrifugation of T, fraction (see Table I) at 100,OOOxg for 60 min, but also the tail fragments (T, fraction) exhibited some enzyme activity. U’ith the Experimental

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Mitochondrinl

functions

of bull spermntozoa

239

possible exception of hexokinase, the enzyme activities found in the midpiece fraction are probably due to a part of tail included in the midpiece. In the same table, the distribution of malic dehydrogenase among the fractions is also presented. This enzyme was distributed almost equally in the midpiece and the tail fractions. A further sonication of the midpiece fraction was necesTABLE

XI. XAD+-linked glycerol-l-phosphate hydro~genuse activities in trail fraction

Baranowski

enzyme

= crystalline

SAD+-linked

dehydrogenase and lactic tlefrom bull spermcctozorr.

glycerol-l-phosphate muscle.

dehydrogenasc

m,umolcs NADH oxidizcd/miil

Glycerol-l-phosphate dehydrogenase activity 0.5 ml of tail frziction tail fraction t dihydroxyacetone phosphate tail fraction ?- dihydroxyacctone phosphate enzyme (0.5 fig) dihydroxyacetone phosphate f I~aranowski (0.5 pg) Lactic 0.05

dchydrogenase activiL> ml of tail fraction

from

rabbit

mpmoles NADH oxidized min/mg protein

3.4 3.1

1.5 1.5

c Baranolvski 13s

60.5

enzyme 13 3

789

180

sary to obtain the full activity of malic dehpdrogenase. The head fraction was practically devoid of these enzymes. Possible source of glycerol-l-phosphrcte in bull spermcrtozou-The failure to demonstrate NM&linked glycerol-l-phosphate dehgdrogenase activity in bull spermatozoa raised the question as to where glycerol-l-phosphate, which could be oxidized intensely by the sperm mitochondria, comes from. It is generally recognized that mammalian spermatozoa preferentially utilize the energy from glycolysis for their motility, provided that they are supplied with fructose in the surrounding seminal plasma [i, 601. Table XIII she\\-s the results of analyses of products from anaerobic fructolysis by washed bull spermatozoa. In agreement with the above results, no significant amount of glycerol-l-phosphate accumulated after anaerobic incubation with fructose, most of the fructose utilized being converted into lactate. Dihydroxyacetone Esperimer~tul

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240

H. Xohri,

T. Mohri

and L. Ernster

phosphate, pyruvate, and glycerol were also not detected. There was a discrepancy’in the balance sheet, in that more than 14 per cent of the fructose was not accounted for by these metabolites. This has previously been described by Rothschild [ 791, who has considered two explanations for this discrepancy: (1) conversion of fructose into glycerol, acetate and CO,; and (2) formation of sorbitol from fructose. The present results exclude the first possibility, leaving the second open to be examined. An alternative pathway providing glycerol-l-phosphate is fat degradation. Mammalian spermatozoa oxidize endogenous phospholipids in the absence XII.

TABLE

Distribution

of some glycolytic enzymes and malic dehydrogenase among bull-sperm fragments. Homogenate pmoles

Head of substrate

Enzyme Hexokinase

3.5

Aldolase

(100)

1.8 (28)

Midpiece

Tail

consumed/min/mg (X)

of protein

5.4

7.0

(28)

(46)

49.4

2.5

36.3

143.2

(100)

(3)

(13)

(76)

Glyceraldehyde-3-phosphate dehydrogenase

16.5 (100)

0.0 (0)

22.9 (25)

52.9 (78)

Laatic

281 (100)

26.3 (5)

374 (17)

792 (87)

141.3 (100)

9.6 (4)

350 (45)

267 (57)

Malic

dehydrogenase

dehydrogenase

TABLE

XIII.

Analysis of products from anaerobic fructolysis by washed bull spermatozoa. 0'

90' ,umoles

Fructose Lactate Glycerol-l-phosphate Dihydroxyacetone Pyruvate

phosphate

Glycerol

Experimental

Cell Research

38

Difference

16.8 0.1 0.0 0.0 0.0

10.5 10.8 0.1 0.0 0.0

0.0

0.0

- 6.3 + 10.7 ‘- 0.1 -

Mitochondrial

functions

of bull spermatozoa

241

of seminal plasma or glycolyzable substrates [35, 46, 47, 551. In addition, mammalian seminal plasma contains glycerylphosphorylcholine, the metabolic fate of which is still not clear [20, 23, 561. Furthermore, intact mammalian spermatozoa can oxidize free glycerol as readily as glycerol-l-phosphate [61]. If glycerol set free from fat metabolism were a substrate for respiration TARLE XIV.

Demonstration

of ylycerokinase

activity

in bull spermatozoa.

Specific activity (unitP/mg of protein) Bull Rat

sperm homogenate liver homogenateb

a The amount of enzyme catalyzing b Data from Bublitz and Kennedy

the formation

0.95 0.61 of 0.1 pmole

of glycerol-l-phosphate/hr.

[9].

of bull spermatozoa, these should contain glycerokinase, because the midpiece fraction could not oxidize free glycerol. The data presented in Table XIV indicate that, indeed, bull spermatozoa possess a rather high glycerokinase activity. DISCUSSION

The results reported in this paper demonstrate that the isolated midpieces from bull sperm exhibit enzymic features which closely resemble those of mitochondria from various animal tissues. Thus, the isolated bull sperm midpieces catalyze the aerobic oxidation of various Krebs cycle metabolites, glutamate, /?-hydroxybutyrate and glycerol-l-phosphate, they carry out oxidative phosphorylation, and exhibit respiratory control, to extents which are usually found with ordinary mitochondria. A further similarity is in the oxidation of external NADH, which requires the addition of either cytochrome c [37, 521 or a lower coenzyme Q homologue [37, 411. This latter feature may be of particular interest since it has been considered [36] that the oxidation of external NADH by isolated mitochondria might be an artefact, due to contamination by sub-mitochondrial particles and/or microsomes. In the case of isolated midpieces this explanation appears to be unlikely, in view of both the low centrifugal force (900 x g) used for the sedimentation of the midpieces, and the clean appearance of the preparations on the electron micrographs. Experimental

Cell Research

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H. Mohri,

T. Mohri

anti 1,. Ernster

The relatively high rate of glycerol-l-phosphate oxidation by the isolated bull sperm midpieces raises the question as to metabolic role of this reaction. In muscle from both insects and mammals, the highly active mitochondrial glycerol-l -phosphate oxidase has been implicated in the “glycerol-l -phosphate cycle”, in conjunction with the cytoplasmic glycerol-l -phosphate dehydrogenase, with the function of transferring hydrogen from cptoplasmic NADH to the mitochondrial respiratory chain [ll, 28, 37, 84, 971. In the bull sperm \ve found no indication for an active cgtoplasmic glycerol-l-phosphate dehydrogenase, and hence, the operation of the “glycerol-l -phosphate cycle” would not seem to be the major function of the midpiece glycerol-lphosphate oxidase. A similar situation is encountered in brain, which exhibits a fairly high mitochondrial glycerol-l -phosphate oxidase activity [43, 83: but is poor in cytoplasmic glycerol-l-phosphate dehydrogenase [al, 961. A plausible function of glycerol-l-phosphate oxidase of the bull spermatozoa may be the oxidation of glycerol-l-phosphate derived from phospholipids, which are well recognized nutrients of the spermatozoa [3,5, 18, 47, 351. Moreover, seminal plasma is known to contain fairly large quantities of glycerylphosphorylcholine [20, 23, 361, which, as recently shown by \\‘hite et al. [93], is split into glycerol-l-phosphate and choline by an enzyme secreted from uterine epithelium. The presence of a high glycerol-l-phosphate oxidase activity together with the occurrence of a likewise high glpcerokinase activity in the bull spermatozoa, as revealed by the present work, also affords a logical explanation of the finding of Mann and \\‘hite [(il 1 that $,I\-cerol stimulates sperm respiration. An important question concerns the mechanism by jvhich the energy provided by the mitochondrial system of the midpiece is transmitted to, and utilized by, different parts of spermatozoa. The present data clearly shov that the energy-generating capacity of the midpiece is amply sufficient to cover the requirements of sperm motility. Ho\v the ATP produced by the midpiece is transferred to the contractile elements of the tail, however, remains an unresolved problem. Active movement of ions has been concluded by Steinbach and I)unham [86] to depend on an activation of the headThese authors have shown that semidpiece portions of the spermatozoa. parated tails haw no power to maintain ionic gradients and concluded that the maintenance of normal conducted flagellnr movement depends on ionic gradients pumped into the tail by the head-midpiece machinery. In Tie\\- of these conclusions, provision of energy for active ion transport may be one of the important functions of the mitochondrial system of the midpiece. Finally, ejaculated spermatozoa haye been shown to carry out incorporation of amino Experimenfal

Cell

Reseurch

38

Mitochondriul

functions

of hull spernmtozotr

acids into protein [4, 51 as well as incorporation cerol into lipids [89, 901. Supply of energy further facet of midpiece function.

243

of acetate, glucose and glpfor these processes may be a

SUMMARY Ejaculated bull spermatozoa were disrupted by sonic oscillation and fractionated by differential centrifugation into a head, a midpiece, and a tail fraction. The midpieces, which consist of mitochondria, showed a wellpreserved structure, and were practically free of other cell components, as judging from electron micrographs. The isolated midpieces catalyzed the aerobic oxidation of various substrates including Krebs-cycle intermediates, glycerol-l-phosphate, glutamate, and /3-hydroxybutyrate. Glycerol-l -phosphate was oxidized at the relatively highest rate both in the absence and presence of added cytochrome c, and \vas converted stoichiometrically into dihydroxyacetone phosphate. among the Krebs-cycle intermediates, pyruvate plus malate gave the highest respiratory rate. The addition of cgtochrome c, however, greatly enhanced the oxidation ol succinate, nearly LIP to the rate with glycerol-l-phosphate in the presence of cytochrome c, but stimulated only slightly the oxidation of pyridine nucleotide-linked substrates. Adequate I’:0 ratios were obtained with most of substrates tested. The stimulation of oxidation of glycerol-l-phosphate and succinate by cytochrome c caused a decrease in the P:O ratio. The respiratory control indexes with pyrurate plus malate and glutamate as substrates \vere around 10, while those \vith glycerol-l-phosphate and succinate were 2&I. The oxidation of glycerol-l -phosphate and succinate \vas inhibited almost completely by antimycin A, but not by rotcnone and amytal, whereas the oxidation of pyruvate plus malate A\-as abolished by all three inhibitors. 2,1-lXnitropheno1 uncoupled respiration from phosphorplation, while oligomycin abolished both tightly-coupled respiration and the accompanying phosphate uptake. For the osidation of citrate and isocitrate the addition of either N=ZI>+ or NADP+ was required. The oxidation rates with these substrates were further increased by adding a catalytic amount of either malate or oxaloacetate. Externally added NADH \vas oxidized only slolvly by the midpiece fraction, but the rate of oxidation was strongly enhanced by the addition of cytochrome c. The cytochrome c-stimulated oxidation was inhibited by both rotenone and antimycin ,4, but was accompanied by practically no phosphoE,rlzerin;enfnl

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H. Mohri,

T. Mohri

and L. Ernster

rylation. Indirect oxidation of external NADH by way of the “glycerol-lphosphate cycle” or “/I-hydroxybutyrate cycle” could not be demonstrated. These features indicate that bull sperm mitochondria possess biochemical properties closely resembling those of mitochondria from other tissues, in spite of their greatly modified structure. The distribution of some enzymes in bull sperm fragments has also been examined. Most of glycolytic enzymes were concentrated in the tail fraction, while malic dehydrogenase was located in both the midpiece and the tail fraction. Anaerobic fructolysis by the bull spermatozoa produced only a minute amount of glycerol-l-phosphate, and most of fructose consumed was converted into lactate. The demonstration of glycerokinase activity in bull sperm homogenate suggests a possible source of glycerol-l-phosphate, which is rapidly oxidized by the midpiece fraction. Our thanks are due to Dr. B. A. Afzelius for his valuable advice in preparing electron micrographs. Thanks are due also to Vet. H. Sandefeldt of the Stockholms Lans Centrala Seminf6rening for his continuous and generous supply of bull semen. REFERENCES 1. AND&, 2. AZZONE, 22,

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Cell Res.

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