COAGULATION AND LIQUEFACTION OF SEMEN*
PRoTEoLYTIC ENZYMES AND CITRATE IN PROSTATIC FLUID
BY CHARLES HUGGINS, M.D., AND WILLIAM NEAL, M.D.
(From the Department of Surgery of The University of Chicago, Chicago)
(Received for publication, August 3, 1942)
The semen of man and animals is delivered from the urethra in a liquid
state but differs thereafter in its properties in three principal ways. In the
guinea pig it becomes an elastic solid which is maintained in this condition
both in vitro and in the vagina for many days. The semen of the dog remains
liquid. The ejaculate of normal man solidifies but then liquefies in a few
minutes at room temperature.
The solidification of the semen of the guinea pig is due to the action of an
enzyme, vesiculase (1, 2) derived from a special region of the prostate (3) on
the proteins of the seminal vesicle. No experiments have been reported deal-
ing with the solid-liquid phases of human semen or with the persistence of the
liquid state of canine semen, matters with which the present paper is con-
Our first observation was that a mixture of human semen with blood under-
went coagulation but soon liquefied. Most of the present experiments deal
with various phases of the effects of semen and its components on blood and
its clotting constituents.
Human semen was obtained by manual ejaculation. Following liquefaction, it
was centrifuged and the supernatant fluid used immediately for testing. Canine
semen was obtained by pilocarpine stimulation following the prostatic isolation pro-
cedure of Huggins, Masina, Eichelberger, and Wharton (4); this fluid was passed
through a Seitz filter to obtain a sterile filtrate free from cells.
Fresh blood and citrated plasma were obtained from various species. The plasma
was procured by centrifugalization of blood drawn into a solution of sodiumcitrate,
5 per cent, of which 1 co. was used for each 9 co. of blood; it was recalcified with a solu-
tion of CaC12, 1.5 per cent, of which 0.33 cc. was used for each cubic centimeter of
Testing for lytic activity was carried out by mixing 1 cc. of blood or plasma with
Icc. of semen or other fluid under examination, either undiluted or diluted to 1 cc. with
saline; after mixing, the tubes were stoppered and placed in a water bath at 37°C. and
* This investigation was supported by grants from the Committee for Research in
Problems of Sex, the National Research Council, and from the National Committee
on Maternal Health.
COAGULATION AND LIQUEFACTION OF SEMEN
the times of coagulation and of subsequent liquefaction, when it occurred, were noted.
In stating the dilutions in this paper the actual content of secretion in 1 ce. is given;
saline refers to an aqueous solution of NaCI, 0.15 molar in concentration, and incuba-
tion refers to 37°C. Where citrated plasma was used the specified concentration of
calcium chloride was added according to two plans; either the plasma was recalcified
immediately after adding the fluid to be tested, or after a delay of minutes to hours to
permit a prolonged contact, in the liquid state, of the plasma and the fluid under test.
As control, saliva, bile, cerebrospinal and spermatocele fluids were substituted for
semen and the effects on the dotting and liquefaction of blood noted.
In testing the effect of the lytic factors in semen on the dotting constituents of
human blood, fibrinogen, thrombin, and thromboplastin were prepared from beef
plasma and lung by the method of Smith, Warner, and Brinkhous (5) and prothrombin
by the technique of Seegers and colleagues (6).
Citrate analyses were made by the method of Pucher, Sherman, and Vickery (7)
and throughout this paper the term, citrate, refers to the combined amount of citric
acid and its salts as determined by this method. Trypsin was determined by the
method of Anson (8).
The opacity developing coincident with clot formation induced by recalcification
of citrated plasma, and the effect of semen upon this density were studied in an Evelyn
photoelectric colorimeter. The colorimeter was mounted in an incubator at 37°C.
and all of the solutions were brought to this temperature before using. A 6600 Ang-
strom light filter was used. The total volume in each colorimeter tube was 13.5 ce.
distributed as follows:--
5 cc. of plasma + 2 cc. of semen (saline in the control tubes) + 5 cc. of saline + 1.5
cc. of CaC12.
Following recalcification, readings of the galvanometer were recorded each 12 sec-
onds until a steady state had been reached following coagulation, or until it was
evident that dotting was not occurring.
The Phenomenon of Spontaneous Lysis of Human Semen.--The semen of
normal men after ejaculation forms a solid mass and the container may be
inverted without losing any of the material; within a few minutes softening
and partial liquefaction is observed and within 15 minutes the ejaculate is in
a liquid state save for a few sago-like particles, derived from the seminal
vesicles, which remain for more than 1 hour.
The lytic phenomenon may be observed well through the microscope.
a typical example, at 3 minutes after ejaculation a thin section appeared as
many interlaced bundles of long, clearly defined, parallel, refractile fibres; at
4 minutes the fibres appeared swollen; at 5 minutes the regularity had disap-
peared and the fibres were arranged haphazardly; at 6 minutes there was
large scale liquefaction with movement visible as many of the fibres were
rapidly breaking up. No fibres were seen 8 minutes after ejaculation.
In a man with hyopgonadism due to undescended testes the semen did not
CHARLES HUGGINS AND WILLIAM NEAL
clot; following intramuscular injection of testosterone propionate, 25 rag.
daily for 15 days, normal clotting was observed.
The Effect of Semen and Prostatic Fluid on the Coagulation Time of Blood.--
The clotting time of whole blood, 1 cc. mixed with saline, 1 cc., in 144 instances
ranged from 3 to 10 minutes.
(a) Human semen: Two effects were observed, shortening and prolongation
of the coagulation time of whole blood. Of 23 seminal specimens tested
against whole human blood, 11 prolonged the coagulation time, while 12
caused a decrease of the clotting time as compared to the control saline mix-
tures. In the group in which a mixture of semen lessened the time of coagula-
tion, the effect was evident with as little semen as 0.025 cc. (Table I). This
Effect of Semen and Other Human Secretions on the Coagulation Time of Human Blood
1 cc. of whole blood + 1 co. of secretion, diluted secretion, or saline.
Nature of secretion
Semen, group 1
Semen, group 2
Prostatic fluid heated
Prostatic fluid and CaCls*
Seminal vesicle secretion
Amount of secretion in test
5 5 --
* Calcium chloride, 1.5 per cent, 0.33 cc.
lessened coagulation time is due to thromboplastic substances in appreciable
amounts. In the group with prolonged clotting with semen in 1 cc. amounts
no, or slight clot, was observed in 30 minutes; in this group, semen in 0.2 or
0.1 cc. amounts often lengthened clotting time to, 15 or more minutes.
(b) Human prostatic fluid: All specimens of undiluted fluid tested (from 18
men) inhibited the dotting of human blood. With prostatic fluid 0.1 cc.,
dotting occurred in 14 to 50 minutes in 5 specimens, while no clot was observed
in 2 hours in 13 observations. The coagulation time of blood mixed with
prostatic fluid 0.02 cc. was approximately normal.
Coagulation of the blood was always induced by adding calcium chloride,
1.5 per cent, 0.33 cc. to prostatic fluid, 1 cc. In 5 cases incoagulability, or
greatly prolonged clotting time, was abolished and a clotting time shorter
than normal supervened; in 3 cases the coagulation time was greatly decreased
COAGULATION AND LIQIrEFACTION OF SE~EN
far greater fibrinogenase activity than human semen. It was readily possible
by dilution to eliminate the weaker activity and to retain the stronger; this
is evidence for the presence of two distinct proteolytic agents acting on fibrin
and fibrinogen respectively. Garner and Tillet (29) observed that "solutions
of human fibrinogen after brief incubation with fibrinolysin lose the capacity
to form thrombin." The observed quantitative and species differences of
proteolysis in semens do not fall in with the interpretation of these workers,
whose observation on streptococcal filtrate with respect to fibrinogen is the
same as ours on dog semen. We conclude that the agent in semen which
liquefies fibrin resembles closely or is identical with the fibrinolysin of Tillett
and Garner, and that the inactivation of fibrinogen is due to a separate agent,
Certain specimens of human semen shorten the coagulation time of whole
blood because of the presence of active thromboplastic agents, while other
samples prolong its coagulation time. Human prostatic fluid in large amounts
always delays or abolishes blood coagulation. The delay or absence of clot-
ting is counteracted by adding calcium ions and is due to the large concentra-
tion of citrate in prostatic fluid and in some semens.
While most specimens of dog semen shorten the coagulation time of blood
because of their thromboplastic activity, certain specimens render blood in-
coagulable or delay coagulation; in contrast to human semen, this adverse
effect on coagulation is not overcome with calcium ions and is due to a dif-
ferent mechanism, the lysis of fibrinogen. The citrate content of dog prostatic
fluid is small.
Human semen which has become liquefied does not contain thrombin or
prothrombin, but fibrinogen and thromboplastic substances are present. Beef
fibrinogen added to semen is destroyed by incubation for 18 hours, but added
prothrombin and thromboplastic substances are still present after this treat-
ment. Dog semen, in some instances, contains small amounts of thrombin.
The semens of man and dog contain a fibrinolysin for human blood which
seems not to differ greatly from the fibrinolysin associated with hemolytic
streptococci. The blood of the donor of prostatic fluid is susceptible to
fibrinolysis by this fluid. However, the blood of persons with some diseases,
is absolutely resistant to the action of seminal fibrinolysin. In how many
diseases this happens has not yet been determined.
The semens of man and dog both contain an agent capable of inactivating
fibrinogen, but in different amounts. This activity may be called fibrinogenase.
Human semen is rich in fibrinolysin, poor in fibrinogenase; dog semen is rich
in fibrinogenase, poor in fibrinolysin. These species differences, together with
the fact that it is easy by appropriate dilution to retain the stronger proteolytic
CHARLES HUGGINS AND WILLIA.M lqEAL
agent and eliminate the weaker one, imply that fibrinolysin and fibrinogenase
are different entities.
Dog semen, and less constantly human semen, contain very small amounts
All of these proteolytic agents derive from the prostate gland; their secre-
tion in prostatic fluid constitutes a hitherto undescribed function for the
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