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 LIQUEFACTION 0~" SEMEN"
but still remained longer than the normal control. In all cases in which semen
caused a prolonged clotting of admixed blood, the addition of the specified
amount of calcium chloride abolished the delay and coagulation occurred more
rapidly than in the control saline-blood mixture.
(c) Dog prostatic fluid: Of 40 specimens of human blood, 1 cc., mixed with
prostatic fluid, 1 cc., the coagulation time was normal or shortened in 32 cases.
In 4 cases clotting did not occur while only slight clot was observed in 13 to 20
minutes with the fluid of 4 dogs in this group. In contrast to the system,
human prostatic secretion-blood, the addition of calcium ions did not initiate
or hasten clotting and the delay was due to a different mechanism, namely, the
disappearance of fibrinogen as it is discussed below.
Citrate Is the Cause of Prolonged Coagulation Time of Mixtures of Blood and
Human Prostatic Fluid or Semen.--It was found that human prostatic fluid
heated in a water bath at 100°C. for 30 minutes still delayed blood coagulation,
while the delay was abolished by adding calcium ions (Table I). Large
amounts of citrate in the prostatic fluid were discovered by Scherst6n (9)
and the finding was confirmed by Dickens (10). We likewise observed large
amounts of this acid, obtaining the following values for citrate in each 100
cc.; 15 specimens of human semen, 140 to 637 rag.; 9 samples of human prostatic
fluid, 480 to 2688 mg.; 20 lots of dog semen, 0. to 2.6 mg.; 2 analyses of human
seminal vesicle secretion, 15 and 22 rag. While Huggins, Scott, and Heinen
(11) found a high calcium content in human semen (6 millimols per liter) and
in human prostatic fluid (30 m~r per liter) the concentration of citrate in
prostatic fluid and some seminal specimens is so high that the calcium ions
are reduced preventing thrombin formation and blood coagulation. It seems
reasonable to conclude that the adverse effect of human prostatic fluid on
blood coagulation is a citrate effect.
Clotting Constituents of Human Semen.--The principal clotting constituents
of blood were prepared from beef plasma and lung. In order to determine
the presence of these or similar substances in semen, various combinations of
them were placed in contact with human semen which had become liquid and
the presence or absence of coagulation and the clotting time determined.
Differences were observed depending on whether calcium ions were added to
induce clotting immediately after adding semen or after preliminary incubation
of semen with the clotting fractions of blood in the liquid state.
(a) Immediate induction of clotting: Human semen was found capable of
substituting for fibrinogen and thromboplastin but not for prothrombin in
inducing clotting (Table II, lines 1-6). The clot formed in the absence of
added fibrinogen, was not firm, indicating that the fibrinogen content of semen
was less than that of the prepared extract. Goldblatt (30) discovered that
human semen contained thromboplastin.
(b) Delayed induction of clotting: Incubation of prothrombin with semen
CHARLES HUGGINS AND WILLIAM NEAL
both for 40 minutes and 18 hours decreased the coagulation time on adding
fibrinogen, thromboplastin, and calcium ions (Table II, lines 7-10).
tion of fibrinogen with semen for 18 hours abolished clot formation on adding
the other principle clotting agents. Neither thromboplastin nor prothrombin
were inactivated by incubation with semen for 18 hours; of the proteins im-
plicated in blood coagulation only fibrinogen was destroyed.
The Effect of Human Semen on the Clotting Constituents of Beef Blood
Fi ri I Throm-
b - Iboplas-
C6. [ CC.
Small amount of clot
A few fibres of fibrin appeared
at 15 sec. Solid clot in 10
The constituents designated * were incubated for 40 minutes at 37°C. and the other
elements were then added.
The constituents designated ~ were incubated for 18 hours and the other elements were
Lyric Action of Human Semen on Normal Human Blood.--The blood of
healthy persons when mixed with certain amounts of human semen or prostatic
fluid, will still clot but subsequently liquefy. With equal concentrations of
blood and semen, lysis occurs in 1 to 5 hours at room temperature, slightly
more rapidly at 37°C. and not at all at 4°C.
saline were mixed with blood, 1 cc., and observed at 18 hours (Table III).
In all instances semen, 0.03 cc., induced lysis; in 4 cases semen, 0.01 co.,
liquefied the clot. Twenty samples of prostatic fluid were studied in the
same way; lysis occurred in all with prostatic fluid, 0.02 cc., in 18 hours, and
in one fluid 0.002 cc. induced lysis.
Human semen did not liquefy beef plasma.
Serial dilutions of semen in
Lysis of dog blood and plasma