Effect of α-chlorohydrin on cauda epididymis and spermatozoa of the rat and general physiological status

EFFECT OF a-CHLOROHYDRIN ON CAUDA EPIDIDYMIS SPERMATOZOA OF THE RAT AND GENERAL PHYSIOLOGICAL P.D.C.

Department University of

AND STATUS

Brown-Woodman I.G. White

of Veterinary Sydney, N.S.W.

Physiology, 2006 Australia

ABSTRACT The effect of a single injection of a high dose (90 mg/Kg) of a-chlorohydrin (3-chloro-1, Z-propanediol) on the metabolic activity of the spermatozoa and tubule of the rat cauda epididymis was investigated. The respiratory activity of the liver and kidney, and glucose metabolism of the latter organ were also studied. Forty-eight and 240 hours after injection, the oxygen uptake and motility of sperm flushed from the cauda was greatly reduced as was the amount of glucose utilized, the amount of glucose oxidized and the accumulation of lactic acid. By contrast, a-chlorohydrin had little effect on the respiratory activity of the flushed epididymal tubule, liver or kidney tissue, but stimulated glycolysis in the kidney. It is concluded that the metabolic effects of a-chlorohydrin are fairly specifically confined to the spermatozoa in the epididymis and this may account for the reduced fertility and motility. At the high dose used, the drug also caused loss of body weight, hypertrophy of the adrenal glands and an increase in the total leukocyte count.

Accepted

for publication

JANUARY

1975

November

VOL.

14,

11 NO. 1

1974

69

CONTRACEPTION

INTRODUCTION There is evidence to implicate the cautia epididymis as a site of action of a-chlorohydrin since the onset of infertility occurs within 3 to 6 days of its administration to rats (1, 2) and the spermatozoa take at least a week to pass through the duct (3). Furthermore, the studies of Crabo (41 indicate that cc-chlorohydrin is concentrated in the cauda region. This study was undertaken to determine whether a-chlorohydrin might affect the metabolism of spermatozoa directly or through disruption of the metabolism of the epididymal tissue in the caudal region of the duct, after a single injection at a high dosage. Since earlier work in this laboratory and studies by Setty Kar (5) indicate that cr-chlorohydrin is toxic to rats, the metabolic activity of liver and kidney tissue was also studied, along with the physiological status of the animals, to determine whether damage was localized to the reproductive tract or of a more general nature.

and

METHODS The epididymis and other organs from a group of control rats were examined and from animals 48 and 240 hours after injecting a-chlorohydrin subcutaneously (90 mg/Kg body weight). For every estimation, both epididymides were dissected from each of 3 mature rats (Spraque-Dawley strain weighing approximately 300 g) anaesthetised with pentobarbitone sodium (Sagatal, May and Baker Ltd. Vie.). The epididymides were immediately placed in calcium-free Krebs Ringer phosphate (KRP, pH 7.3). Material from 3 epididymides was pooled for study of the metabolic activity of the spermatozoa and the 3 contralateral epididymides used for assessing the metabolic activity of the epididymal tubule. Lengths of the tubule were taken from the shaded area of the cauda epididymis shown in Fig lc. Spermatozoa Epididymal tubules were cut into lengths about one cm long, under a dissecting microscope. The contents of about 5 such lengths from one epididymis from each rat were gently squeezed into 0.2 ml calciumfree KRP. The final 0.6 ml obtained from the 3 epididymides was centrifuged for 5 minutes at 1000 rpm, the supernatant discarded and the plug of spermatozoa made up to 1.4 ml with calcium-free KRP. An 0.5 ml aliquot of this suspension of spermatozoa was added to a Warburg flask containing 0.5 ml calcium-free KRP, 0.5 mg each of crystalline penicillin G any4 streptomycin sulphate, and 7 umoles iI(+) glucose and 0.25 pCi D-[UC] glucose/ml. A further 0.5 ml of the suspension of spermatozoa was added to 0.5 ml of diluent and deproteinized with 5% ZnSOq.7H20 and 0.3 N Ba(OH12.8H20 for determination of initial glucose and lactic acid concentration. The Warburg flasks were incubated at 37’C for 3 hours and shaken at 60 cycles per minute. Oxygen uptake was measured by the direct manometric method described by Umbreit, Burris and Stauffer (6). wi gas phase. The amount of glucose oxidized was calculated from collected in 0.5 ml 20% KOH (w/v) in the centre well of the Warburg2flask. Radioactivity was determined in a Nuclear Chicago scintillation counter.

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1975 VOL. 11 NO. 1

CONTRACEPTION

the samples were deproteinized as described After incubation, Initial and final glucose concentrations were determined before. Lact i c enzymically and the amount of utilized glucose calculated (7, 8). acid accumulation was also measured enzymically (9). Duplicate 0.1 ml samples of the suspension of spermatozoa were added to 2.0 ml formal saline and the number of spermatozoa counted in a haemocytometer. A drop of the suspension of spermatozoa was added to 0.3 ml KPP containing 300 mg% fructose for scoring of motility with by the system of Emmens (IO). There was no evidence of conby epithelial cells.

calcium-free a microscope tamination Tubules

Short lengths (approximately one cm) of epididymal tubule were dissected free of connective tissue in a watch glass containing warm (37’C) calcium-free KRP. A glass microcapillary with the tip drawn out to approxj,mately 0.05 mm, was connected to a syringe via metal and plastic tubing. The metal tubing was clamped in a micromanipulator (Prior, England) and the system filled with warm calcium-free KRP. The tip of the capillary was inserted into one end of a length of tubule and the contents flushed out. The flushed tubule was then washed in calcium-free KRP and the excess liquid blotted off. The lengths of tubule for one estimation (about 60-90 mg) were weighed in a vial containing 0.2 ml calcium-free KRP. The tubules were again blotted and placed in I.0 ml diluent in the outer compartment of a Warburg flask. The diluent contained 4 umoles DC+) glucose per ml and 0.25 uCi D-[U-14C] glucose per ml and antibiotics. Oxygen uptake, glucose metabolism and lactic acid accumulation were measured as described for the spermatozoa. Other

Organs

and Blood Liver slices imately one mm square, and incubated according idymal tubule. Oxygen as previously indicated.

recorded procedures.

The weights and leucocyte

and slices of kidney cortex and medulla, approxwere weighed in 0.2 ml calcium-free KRP, blotted to the method described for the lengths of epiduptake and other metabolic parameters were measured

of the counts

paired adrenal performed using

glands, thymus a haemocytometer

and

spleen were and standard

Statistical

Analysis The results in Tables I and II have been subject to standard analysis of variance and treatment means compared with the control by a “t” test using the interaction mean square to calculate the standard error of the difference between the pair of means.

RESULTS Spermatozoa

from Cauda Epididymis Oxygen uptake, glucose oxidation, glucose utilization and lactic acid accumulation were all reduced 48 and 240 hours after injecting a-chlorohydrin (Fig la and Table I). Motility of the spermatozoa was also signiticantly decreased (4.6 at 0 hours, 1.9 after 48 hours and 2.0 after 240

JANUARY

1975 VOL. 11 NO. 1

71

it

per

II

are

,,

the

!I

0 hr

number

from

spermatozoa. of

+ 0.388 k +

I .930 2.816 2.268

+

i

2.080

< 0.05

observations.

13.8

6.7

13.8

5.1

***

Mean

ir 0.192

1.835

?‘i

i

2.445

2.3*

4.9

Significantly

S.E.

0.293

per

0.109””

0.103

0.271

0.135**

5

0.278

I .8***

0.335

f + 0.179”

1.372 0.702

1.9***

1 I .6

100

a-CHLOROHYDRIN

Glucose Utilized (umoles)

FROM

I

Uptake

OF TISSUES

P < 0.01

P

+

160.3

48

240

+

68.9

240

+

t

66.6

5

f

82.1

0

48

177.5

+

14.5

240

159.4

r

0

*

(PI

12.1

different

I08

Oxygen

26.1

(hr)

0

after

ACTIVITY

48

Time Injection

parenthesis

Significantly

**

t

1-T

S.E.

*

f

in

Mean

Figures

i

(8)

Kidney

epididymis (5)

cauda

Tubule

from

cauda

epididymis (5)

from

Spermatozoa

Material

METABOLIC

TABLE

0.021

t 0.285

different

from

0.028

mg tissue.

0.030

f t

0 hr

I

2.442

P < 0.001

t t

1.293 2.615

r

1.847 0.014**

t

0.174

0.295

5

2.347

0.008***

*

0.156

0.367

i: 0.242

2.136

0.017

f

0.264

0.217**

0.368**

0.151

0.236

0.114

+ 0.094

0.019

+ 0.090

0.018

‘r 0.014

0.069 o.ooo*

0.013

T 0.005*

Acid (umoles:

+

Lactic Accumulated

0.007

Oxidized es)

RATS

0.056

Glucose f~mol

TREATED

CONTRACEPTION

hours). Tubules

from Cauda Epididymis The oxygen uptake of isolated tubules from the cauda epididymis was only slightly reduced 48 and 240 hours after injecting a-chlorohydrin (Fig lb and Table I) and the effect was not as great as seen in the spermNo significant change occurred in glucose utilization or lactic atozoa. there was a highly significant acid accumulation (Table I); however, reduction in the amount of glucose oxidized by the tubules. Kidney

and

Liver The oxygen uptake of liver (Fig 2a) and kidney (Fig 2b and Table I) slices was not significantly altered after injecting a-chlorohydrin. there was a significant increase in the amount of glucose utilized However, and lactic acid accumulated by kidney slices prepared from rats after There was no significant difference injecting a-chlorohydrin (Table I). in the amount of glucose oxidized by kidney tissue after injecting a-chlorohvdrin. Organ

weights

after change

The weight of the adrenal glands was increased injecting a-chlorohydrin (Table II) but there was in the weight of the spleen or thymus (Table II). TABLE

Organ

.Adrena

Contro

Is

(2)

40.1

+

(mg)

I

hours

48

55.8

-I

240

3.7**

52.9

hours

*

3.7”’

Spleen

828.1

+ 48.12

923.6

+ 54.9

163.4

+ 53.0

Thymus

319.6

k

348.4

+

312.9

+ 25.8

21.3

Values represent ** Significantly White

Blood

Body

Weight

hours

II

ORGAN WEIGHTS

2.4

48 and 240 no significant

mean of different

I5

15.2

observations than 0 hr

P < 0.01

Cell Count The number of leucocvtes was areater in blood samples taken 48 (10758/cu mm) and 240 (12075/cu’mm) hour; after injecting cr-‘chlorohydr ,i n compared with control samples (8872/cu mm). Values represented mean o‘f I5 observations.

body weight weights at respectively,

Paired “t” tests showed a highly significant reduction in the of rats following the injecTion of-a-chlorohydrin. The mean 48 and 240 hours were 346 and 324 g compared to 350 and 330 g, prior to injection (n = 15).

JANUARY 1975 VOL. 11 NO. 1

73

Figure

I

x 0/

i

9

/

GO

120 time

180

Oxygen uptake of spermatozoa of wchlorohydrin (90 mg/Kg)

0f 0

O/O

)y+”

x/

x/’

spermatozoa

I, , k

-/

X

a epididymal

of

0

and tubule n = 5.

incubation

s

x” ::

12

from

(min)

cauda

b epididymal

epididymis

tubule

from

rats

before

and

after

injection

& -chlorohydrin

o 240 hours after injection

after

epididymis

cpididymis

cpididymis

L -chlorohydrin

48 hours

control

cauda

corpus

caput

injection

l

x pre-injection

c

CONTRACEPTION

(an%!&

JANUARY

~a!)

6woo(/j~)

1975 VOL. 11 NO. 1

owdn

u&h~

75

CONTRACEPTION

DISCUSSION Very little of the glucose utilized by the epididymal spermatozoa of the rat could be accounted for by conversion to lactic acid or by oxidation. In this respect, the epididymal spermatozoa of the rat are quite different from those of the ram and bull and seem to have a closer metabolic affinity to the testicular spermatozoa of these species, which convert a higher proportion of glucose to intracellular lipid or inositol (11, 12). Forty-eight hours after injecting a-chlorohydrin, there was a highly significant decrease in all metabolic parameters of the rat spermatozoa and since the spermatozoa take at least 7 days to pass along from the testes (3), the site of action in the present experiments must be in the cauda epididymis. Such a conclusion is amply confirmed by experiments on the ram (13, 14, 15, 16) which indicate that a-chlorohydrin inhibits the enzyme glyceraldehyde phosphate dehydrogenase in spermatozoa. Samojlik and Chang (2) have also found that a-chlorohydrin decreased the oxygen uptake of rat spermatozoa recovered from the epididymis and the reduction in metabolic activity of the spermatozoa in our experiments was accompanied by a 50% decrease in motility. A similar relation between infertility and depressed spermatozoan metabolism and motility has also been observed after injecting rams with a-chlorohydrin (13, 14) and inhibition of metabolism may well be the prime action of a-chlorohydrin. The metabolic pattern of the tubule from the cauda epididymis of the rat clearly differs from that of the spermatozoa, and much of the utilized glucose can be accounted for in terms of lactic acid and carbon dioxide. The metabolism of the tubule is much less sensitive to injected a-chlorohydrin; thus the oxygen uptake and amounts of glucose utilized and converted to lactic acid were little affected although there was some reduction in glucose oxidation. There were no detrimental effects on the metabolism of liver and kidney slices. of kidney slices was considerably enhanced. experiments that the derangement of metabolism n-chlorohydrin is confined to the reproductive the epididymis, it is the spermatozoa rather most affected.

of

injected a-chlorohydrin In fact, the glycolysis It appears from these caused by injected tract and even within than the tubule that are

The dose of a-chlorohydrin used in the present experiments is considerably greater than the minimum required to reduce fertility (21) and a number of rats died. A post-mortem examination revealed haemolytic gastroenteritis as the cause of death, probably precipitated by the a-chlorohydrin. The decreases in food and water intake, increase in adrenal weight and leukocytosis are all probably due to the non-specific action of the high dosage of a-chlorohydrin. Such phenomena reflect the general response of the body to toxic

76

JANUARY 1975

VOL. 11 NO. 1

CONTRACEPTION

stimuli (17, 18, 19, 20) and may It is not implied that the drug. or responsible for, the antifertility

not be elicited by lower dosages of such effects are associated with, action of a-chlorohydrin.

ACKNOWLEDGEMENT

for his supported indebted

The authors would like to thank Professor C. W. Emmens interest and advice throughout the study. The work has been We are financially by the World Health Organisation. for the gift of u-chlorohydrin. to the Upjohn Company, Kalamazoo,

REFERENCES 1.

Coppola, J. A. An extragonadal 8: 43-48 (1969).

2.

Samojlik. 2-propanediol

3.

E.

Risley, P. “Mechanisms New York,

and Chang, M. C. (U-5897) on rats.

L. Physiology Concerned with 73-133 (1963).

male

antifertility

Antifertility Biol.

of the male Conception”.

Reprod.

Crabo, B. and Appelgren, L. E. Distribution in mice and rats. .I. Reprod. Fert. 30:

5.

Setty, B. S. 2-propanediol. J. Exp. Viol.

6.

Umbreit, W. W., Burris, Biochemical Techniques.

7.

Schmidt, Harnzucker klassischen

F.

Kar, A. B. Nature An Oral antifertility 8: 49-50 (19701.

activity 2:

accessory Ed. C.

4.

and

agent.

of 161-163

and site agent

R. H. and Stauffer, Burgess Publishing

of for

Life

Sci.

of 3-chloro-1, 299-304 (19701.

organs. In G. Hartman.

Macmillan.

[14C] cr-chlorohydrin (1972). action of the male.

3-chloro-1, Indian

J. F. Manometric Co, Minneapolis

H. Enzymatische Method zur Bestimmung von Blut-und unter Beriicksichfigung von Vergleichsuntersuchungen Methoden. Internist 4: 554-559 (1963).

and (1972).

mit

a.

Werner, W., Rey, H. G. and Wei linger, H. On the properties of a new chromogen for the determination of glucose in the blood according to the GOD/POD method. Z. Analyst. Chem. 252: 224-228 (1970).

9.

Barker, lactic

J. N. acid.

JANUARY 1975

and Britton, J. Physiol.,

H. G. Lond.

VOL. 11 NO. 1

The 138:

enzymic estimation 3-4~ (1957).

of

L (+I

77

CONTRACEPTION

78

10.

Emmens, C. W. The motility and viability at different hydrogen ion concentrations. 106: 471-481 (1947).

11.

Scott, T. W., Voglmayr, J. K. and Setchell, B. P. Lipid composition and metabolism in testicular and ejaculated ram spermatozoa. Biochem. J. 102: 446-461 (1967).

12.

Voglmayr, J. K. and White, I. G. Synthesis and metabolism of myoinositol in testicular and ejaculated spermatozoa of the ram. J. Reprod. Fert. 24: 29-37 (1971).

13.

Brown, P. Cl. C. and White, I. G. Studies on the male antifertility drug 3-chloro-1, Z-propanediol. J. Reprod. Fert. 32, 337-338 (1973).

14.

Brown-Woodman, P. D. C., Salamon, S. and White, I. G. Effect a-chlorohydrin on the fertility of rams. Acta Europaea Fertilitatis (1974) (in press).

15.

Brown-Woodman. P. D. C., White, I. G. and Salatron, S. The effect of a-chlorohydrin on the fertility of rams and on the meta6olism J. Reprod. Fert. (19751 (in press). of spermatozoa in vitro.

16.

Suter, D. A. I., Brown-Woodman, P. D. C., Mohrl, H. and White, The molecular site of action of the anti-fertility agent a-chlorohydrin in ram spermatozoa. J. Reprod. Fert. (1975) (in press).

17.

The general adaptation syndrome and the diseases Selye, H. J. Clin. Endocr. Metab. 6: 117-230 (19461. adaptation.

18.

Selye,

19.

Fifth annual Selye, H. and Heuser, G. Publications, Inc.,New York (1955).

20.

White, sheep.

21.

compounds: Ericsson, R. J. and Baker, V. F. Male antifertility biological properties of U-5897 and U-15,646. J. Reprod. Fert. 21: 267-273 (1970).

H.

Studies

on adaptation.

of rabbit spermatozoa J. Physiol., Lond.

Endocrinology report

syndrome

I. G.

of

169-188

on stress.

I. G. Blood changes during the adaptation J. Endocr. 7: 143-153 (1951).

JANUARY

21:

of

(1937).

M. Cl.

in

1975 VOL. 11 NO. 1