Secretory Function of Male Accessory
Organs of Reproduction
in Mammals
THADDEUS
From
MANN
the Molteno
University
and CECILIA
Institute
LUTWAK-MANN
and the Biochemical
of Cambridge,
Laboratory
Cambridge, England
of reproduction in mammals comprise several
glandular structures concerned with the production of certain specific and
highly complex secretions, which together constitute seminal plasma, the
medium and vehicle for spermatozoa. In most mammalian speciesthe bulk of the
accessorygland secretionscomesfrom the prostate, seminalvesicle, epididymis and
bulbourethral gland or Cowper’s gland; the contribution of the remaining glands is
comparatively small.
Until not a very long time ago the secretory function of the male sex organswas
largely unknown; it required much scientific effort to overthrow the conviction
firmly entrenched in somequarters that, for example, the vesiculaseminalisis not a
mere receptaculumsemi&, or a burial-ground for aged spermatozoa,but that on the
contrary, it is a gland endowedwith a characteristic secretory function (42, 77, 108,
I 20, 153, I 73, I 78). Progressin this branch of physiology was slow owing mainly to
lack of information about the chemical nature of the various secretions.However, in
recent years there was a great move forward when at last several substanceshad
been discovered and identified in the secretionsof the prostate and seminal vesicle,
such as citric acid by Schersten in 1929 (201), prostatic phosphataseby Kutscher
and Wolbergs in 1935 (II~),
fructose by Mann in 1945 (142) and phosphorylcholine
by Lundquist in 1946 (I 24). But even now analytical studiesremain often limited to
the seminal plasma as a whole, and investigations upon the individual secretions
continue few. A notable exception is the work on prostatic secretion of man and dog,
reviewed in the “Physiological Reviews” a short time ago by Huggins (80).
HE MALE ACCESSORY ORGANS
CHARACTERISTIC
FEATURES OF THE SECRETORY FUNCTION OF MALE
ACCESSORY ORGANS
Chemical Constituents. The secretions of the male accessoryglands differ in
several ways from other body fluids. Apart from a high concentration of fructose,
citric acid, phosphorylcholine and phosphatase, they were found to contain some
unusual proteins (64, 195) and lipids (203), amylolytic and proteolytic enzymes
(59, 87) and /3-glucuronidase(214); the mineral composition also is known to be
different from other body fluids. The high calcium content of the human prostatic
secretion is generally regarded as responsiblefor the deposition of the so-called
prostatic calculi (82) which consist chiefly of calcium and magnesiumphosphate but
include also cholesterol (7553, citrate (2.3%) and protein (8%). A deposition of
crystalline ammonium urate was reported recently in the semenfrom a bull with
testicular hypoplasia (8); this, however, remains an isolated statement at present.
27
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Prostatic calculi and the closely related but much softer bodiesknown as coypoya
amyzacea, both develop probably from desquamated cells and prostatic secretion.
They are highly characteristic of the human prostate (168, 169) but are also found
elsewhere,particularly among insectivores (45, 77, 154) where their production is
consideredto be one of the chief secretory functions of the prostate gland. Cellular
in origin are probably also the granules found in the accessorysecretionsof rabbit.
In this connection one may recall the early observation of Stilling (2 I I) relating to
the changed appearance of secretory epithelia in rabbit accessory organs after
copulation. There is much evidence that even under normal conditions secretory
processesin male accessoryglands are accompaniedby definite changesin epithelial
structure, varying from desquamationto cell rupture. It follows that resultsobtained
by analysis of secretory fluids, particularly with regard to large molecular substances
like enzymes, do not necessarily reflect the secretion by intact cells alone but may
also be the outcome of cellular break-up. Perhaps these circumstancesshould be
taken into consideration to account for the differences in the chemical character of
the so-called ‘resting’ and ‘stimulated’ secretion.
The ‘Resting’ and ‘Stimulated’ Secretion. A correct assessment
of the output of
fluid from the accessoryorgans can only be made by allowing for the fact that small
amounts are discharged even during intervals between ejaculations, usually in the
urine, contributing the so-called‘resting’ secretion, as distinct from the much larger
‘active’ or ‘stimulated’ secretion which results from sexual excitation, hormone
treatment or the application of certain nervous stimulants. The distinction between
the two types of secretion is particularly important in the caseof the prostate, much
lessso, probably, in the caseof seminalvesicles. Huggins and his colleagues(IO, 80,
81, 83, 204), whosestudieshave produced much valuable information concerning the
two types of secretionin the prostate gland, assess
the resting secretion of the prostate
in man at about 0.5 to 2 ml. fluid per day, and in dog at 0.1 to 2 ml. per hour. By
injecting dogs with pilocarpine hydrochloride, they were able to achieve a state of
stimulation in which the canine prostate was discharging in one hour as much as 60
ml. fluid, i.e. several times the weight of the organ. Stimulation of hypogastric nerves
and nervi erigentes, and application of epinephrine, nicotine and acetylcholine have
all been shown to lead to increasedprostatic secretion in dog (54, 55).
R8le in Reproduction. Doubts have been expressedon many occasionsas to
whether the male accessory organs have a definite rble to fulfil in reproduction,
particularly since in some animals such as guinea pig or rabbit it was possible to
induce pregnancy by insemination with artificially preparedsuspensionsof epididymal
spermatozoa. However, such experiments bear no direct relation to the mechanism
of the natural mating processwhich could hardly operate without the provision of
seminalplasma as an efficient diluent and vehicle for the thick massof closely packed
epididymal spermatozoa. Furthermore, this experimental procedure fails to take
into account the distinct stimulating effect which the accessorysecretionexerts on the
motility of spermatozoaor the fact, establishedby the more recent biochemical research, that spermatozoa derive from the seminal plasma fructose, an important
nutrient which suppliesmetabolic energy for sperm motility and survival (28-30,
48,49, go, 144-146,148). Nor indeedhas it ever beenshown that the sameconception
Janzdary rgp
MALE
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rate can be achieved with equal numbers of artificially inseminated epididymal spermatozoa and normally ejaculated spermatozoa.
Steinach’sand Walker’s early experiments (209, 217, 218) showing that mating,
and to someextent also fecundity, is still possiblein rats after an excision of either
the prostate or the seminalvesicles(though not any more after a total extirpation of
both glands) have often been cited in the past as evidence against the so-calledessentiality of the accessoryorgans; this in spite of repeated warnings by several investigators including Ivanov (97) that the natural usefulnessof accessorysecretionsshould
not be confusedwith the question of their indispensability. At that time the possibility
was never envisaged that in rat the prostate and seminalvesicle may up to a point
replace each other. Although at first sight sucha possibility appearsrather remote, yet
recent biochemical studies on the role of seminalplasmain the nutrition of spermatozoa, and especially the researchesconcerning the distribution of fructose in rat
accessory organs (gsl 96, 150, 152), have shown that this sugar is produced in rat
by the prostate (dorsal lobe) as well as by the so-called coagulating gland which in
Steinach’s and Walker’s early experiments was still regarded as an integral part of
the seminalvesicle (only years later did Walker (219, 220) succeedin showingthat the
coagulating gland is an organ anatomically distinct from the seminalvesicle).
On the other hand, biochemical studies showedin several instancesthat organs
previously regarded as anatomically or even functionally ‘homologous’on the basis
of similar embryonic origin or analogousmorphological structure actually differ in
their chemical secretory activity. While, for instance, in rat the dorsal prostate was
found to secretefructose, the ventral and also the lateral lobe are concernedwith the
elaboration of citric acid (95, 150, 184). Another instructive example is the human
prostate. Morphologically this organ may appear uniform; chemically it is distinguished by the presence, in high concentrations, of citric acid, phosphatase, and
lipid material, consisting chiefly of cholesterol (213) and cephalin (203). This lipid
material, which appears also in the prostatic secretion in the form of ‘lipid bodies’
(57), occurs in all regionsof the prostate gland, and as it stains characteristically red
with eosin it can be studied parallel with the cell structure of the gland. It was
recently found by Huggins and Webster (94) that, following intramuscular injections
of diethylstilbestrol, the cells of the anterior region of the human prostate regress,
and losetheir eosin-staininglipid material at a much higher rate than the corresponding cellsof the posterior region. Thus, in spite of apparent uniformity, there appearsin
the human prostate a certain multiplicity of reaction, at any rate in sofar as concerns
the responseto hormone administration. In their paper the authors point out that
the peculiar functional duality of the human prostate may be not without connection
with the known predilection of the prostatic carcinoma for the posterior region of the
gland (I 23). They also call attention to the fact that there is a striking similarity in
appearance between the estrogen-induced involutionary changes in the anterior
region of a normal prostate and the changesevoked by antiandrogens in the cancer
of the posterior lobe.
Significance of Species Variations and Individual Differences. The secretory
output of male accessory glands depends chiefly on the size of the organs, their
capacity for fluid storageand their secretory ability, i.e. the rate at which the glands
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generate their secretions.All three factors are subject to considerablevariations not
only from one speciesto another but also very markedly between individuals within
one species.Complete absenceof the seminalvesicles in cat and dog, the very large
seminal vesicles and, at the same time, a small prostate in bull, the prominent
Cowper’s gland and absence of other accessory organs in monotremes and some
marsupials,are a few instancesof the striking speciesdifferencesin the male accessory
apparatus of mammals. Man provides perhaps the most outstanding example of
individual variations in the size of the accessory organs, particularly the seminal
vesicles.But the storage capacity of the human vesiclesis on the whole rather small
as compared with speciessuch as bull, ram or boar. In bull it is not excessivefor the
seminalvesiclesto store 50 ml. of secretion, a quantity sufficient to provide fluid for
no less than a dozen ejaculates; this makes possible the collection from a bull of
several consecutive ejaculateswithin a short time (eight ejaculatesin one hour is not
unusual), with but little volume variation between the first and the last ejaculate. In
man, on the other hand, one or two ejaculations usually deplete the vesiclesto such
an extent that a rest of at least two days is required before the glands fully restore
their storage capacity and provide sufficient fluid for a normal ejaculate. In boar the
seminal vesicles and bulbourethral glands rank as the largest among the domestic
animals; here the volume of secretioncontributed by the accessoryglands may reach
half a liter per singleejaculate. However, this large amount of fluid is not discharged
all at once but is voided gradually over a considerableperiod of time; a complete
ejaculation in boar may take as long as 30 minutes. To some extent conditions in
stallion resemblethose in boar, although in stallion the ejaculation doesnot always
proceed to completion, and consequently the time and total volume of fluid produced
at ejaculation are subject to considerablevariations. In contrast to boar the accessory
secretionsin bull and ram are ejaculated almost instantaneously. Man occupiesin
this respect an intermediate position in as much as it is possible to collect several
separatefractions of a single ejaculate according to time of delivery from the urethra
by meansof the so-called ‘split ejaculate method.’ Under such conditions it will be
found that the secretion from the prostate and seminal vesicle is not distributed
equally over the whole ejaculate, but that the prostatic secretion predominates
largely in the early and the vesicular secretion in the terminal fraction.
Dependence of the Secretory Function Upon the Male Sex Hormone. Among
several factors which influence the male reproductive glands the testicular hormone
ranks high in importance. It is well known that the weight, size, histological appearance and secretory output of most accessoryorgansare strictly dependent upon, and
regulated by, the internal secretion of the testes.The typical grossmorphological and
the retrogressive histological changeswhich take place in the accessoryglands after
castration can be counteracted by administration of testicular hormone; in the past
twenty years several so-called‘hormone-indicator tests’ have been elaborated on this
basis,-and somefound a wide practical application in laboratory assaysof the male
sex hormone (12, 36, IIO, 121, 122, 160, 161, 163, 179, 183, 193, 226). A detailed
account of the comparative value of the various tests was given by Moore (160).
More recently two chemical hormone-indicator tests have been developed, the
‘fructose test’ and ‘citric acid test,’ basedon the finding by Mann and Parsons (151,
January
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ORGANS
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I 52) that a direct quantitative
relationship exists between the dose of testosterone
injected into castrated rats and the level of fructose and citric acid production in the
accessory organs. To maintain in castrated animals an active secretion by accessory
organs a daily injection of about one mg. testosterone propionate is required for
rabbit and about 0.1 mg. for rat. Much less, however, is needed if testosterone is
applied directly to the accessory organs (207). It was shown by Lacassagne and Reynaud (I 14) that as little as 2.5 pg. testosterone propionate is sufficient to produce a
response from the secretory epithelium if introduced directly into the rat seminal
vesicle. According to Demuth (41) a response can also be elicited with testosterone
added to a tissue culture of rat seminal vesicle in vitro; with 20 pg. hormone he observed enlargement of individual cells, which assumed an area of 85 X 10~~ mm.2 as
compared with 45 X IO-~ mm.2 in the control culture.
Estrogens have on the whole an inhibitory action on the secretory activity of
male sex organs as shown, for example, by the reduced volume of prostatic secretion
in dog (83) and the lowered level of seminal fructose in rabbit (176) after injections
of diethylstilbestrol.
Much depends, however, on experimental conditions, hormone
dosage and on other factors. Histological studies indicate that a dose of estrogen
sufficient to curtail the secretory activity may nevertheless stimulate the fibromuscular tissue of a male accessory organ and thus cause an enlargement of the
gland (33, 175). Moreover, an estrogenic hormone which inhibits the male gland
secretion, if administered in large doses, may have a definite stimulating effect when
applied in small quantities. The problem of estrogen activity in relation to male
accessory glands has attracted a great deal of attention in recent years from several
investigators and has been adequately reviewed by Emmens and Parkes (47), Zuckerman (225),
Burrows (22) and Bern (12).
The endocrine action of the testes is closely integrated with that of the anterior
pituitary gland. Consequently hypophysectomy,
like gonadectomy, leads to a fall in
the secretory function of the male accessory organs. However, by means of
gonadotrophin preparations it is possible to restore in the hypophysectomized
male
the secretory activity as efficiently as with testicular hormone.
EXPERIMENTAL
TECHNIQUES
Collection of Secretions. The artificial vagina is the most widely recognized
method of semencollection in veterinary practice but it is not applicable on the
whole to separatecollection of secretionsfrom the different glands except, perhaps,
in boar where ejaculatescan be collected in several fractions. However, by means of
this method very valuable information can be gathered provided it is followed by
chemical estimations in the ejaculate of such substancesas citric acid, fructose or
acid phosphatase which are known to be secreted in different parts of the male
accessorysystem and can thus serve as indicators of the secretory function of specific
organs. The artificial vagina can also beusedsatisfactorily for the collection of seminal
fmid from vasectomized or gonadectomizedanimals. In this way, without sacrificing
the experimental animals, Mann and Parsons(I 51) were able to demonstrate that in
rabbits fructose disappears from semen after castration and that it reappears
promptly after subcutaneousinjection or implantation of testosterone.
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31
Electric stimulation is another method of collection which has been successfully
used in several animal species. The electric stimulus can be applied either through
electrodes suitably placed on the body as, for instance, for the purpose of semen
collection in ram (67, 68) and guinea pig (9, 164) or it can be applied directly to the
nerves which supply the accessory organs as was done in the early experiments on
dogs by Eckhard (46) who ligatured the neck of the bladder to prevent the passage
of urine, then placed a cannula in the urethra and obtained prostatic secretion by
electric stimulation of rtervi erigentes. E&hard’s technique was later improved and
developed by Farrell (53).
A permanent separation of the prostate from the bladder by surgical meansand
cannulation forms the basis of the method developed by Huggins and his colleagues
(86) for the purpose of collecting the prostatic secretion from dogs; by this method
pure prostatic fluid can be collected quantitatively from the sameanimal for periods
of months or years.
Vascular perfusion is yet another experimental technique which wasusedrecently
in the study of prostatic secretion (78, 79). On the question of blood supply and
innervation of the prostate and other accessoryorgans the reader is referred to the
articles by Bacq (4), Barrington (s), Farrell and Lyman (54, 55) and Huggins (80,
81)
in which the older literature is also cited, including the pioneer investigations
by Remy, Langley and others.
The methods mentioned above apply to collection of material from living
animals. For many purposes,however, including the quantitative assay of the male
sex hormone in rats by means of the ‘fructose test’ or ‘citric acid test’ (152)~ the
secretionsare obtained from the accessoryglands after removal of these organsfrom
freshly killed animals.
In man the ‘split ejaculate method’ provides oneway for collection of the prostatic
and vesicular secretions(85, 127, 185), while another such method consistsin digital
expressionof the prostatic and vesicular secretion by manual massage,through the
rectum, of the appropriate regions (89).
Assessment of Secretory Activity by Means of Histological and Chemical
Methods. The evaluation of secretory activity in accessory glands by means of
histological methods is based on the observation of certain structures such as, e.g.,
the secretiongranulesin the secretory cells,which are clearly demonstrablein actively
secreting epithelium but which disappear following castration and reappear in responseto specmchormonal treatment. These methods have yielded in the past much
valuable information concerning hormonal effects in rat (162, 165, 166) and rabbit
(12, 119); however, they are applicable to the glands as such, and are of no avail in
the analysis of the secretedmaterial.
In this respect the advantages of the chemicalapproach are obvious. By measuring the content of fructose or citric acid or the activity of phosphatase,one can
arrive at an accurate estimate of the secretory function, often without sacrificing the
experimental animal. The chemical assayscan be carried out repeatedly at chosen
intervals on samplesfrom the sameindividual, and require relatively little time and
material. Thus, for instance, fructose and citric acid can be analyzed accurately in
0.2 ml. seminalplasma of most species,including man.
January Igp
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Transplantation Technique. The studies on subcutaneousand intraocular transplants of sexaccessorytissuerepresenta lasting contribution in the field of physiology
of reproduction; through them several fundamental observations have been made,
particularly as regards the mechanismof hormone activity on the accessoryorgans
(III,
170, 180, 181). A most recent exploitation of the technique of subcutaneous
transplantation was its application to studies on the metabolism of accessorygland
tissuesreferred to in the section on HORMONE-INDUCED SECRETORY
PROCESSES
IN
TRANSPLANTS
FROM
MALE ACCESSORY GLANDS
OF REPRODUCTION.
The procedure
for subcutaneousgrafting, as usedby Price (180, I&) in rats, consistsessentially in
dissection, from newly born or a few weeksold donors, of minute fragments of tissue
from the coagulating gland, seminalvesicle, ventral prostate or dorsolateral prostate,
and insertion of the tissueinto subcutaneoussiteson the abdomenof sexually mature
hosts. As a routine, six or more piecesof donor tissue,each weighing one mg. or less,
are transplanted into a single host in two rows, one on each side of the abdomen.
The subcutaneoustransplant is allowed to grow for several weeks or months. At
autopsy it is found attached to the skin or abdominal wall from which it can easily
be dissected.The weight of such a graft varies greatly according to growth period
and treatment but under optimal conditions as much as one gm. tissue can be obtained. Inside the graft there is an accumulation of secretory fluid which can be used
for chemical analysis (130, I 50, 184).
FRUCTOSE IN MALE
ACCESSORY ORGANS
Fructose in Semen. Since the early researcheson mammalian semenit has been
known that the semenof several mammalian species,including man, contains a
reducing and yeast-fermentable sugar at a concentration exceeding by far that of
glucose in blood. In the extensive literature concerned with seminal sugar it was
mostly either assumedto be glucoseor describedas the reducing sugar of semen(IS,
35, 59, 85, 89, 98, 105, 131-133,
198, Igg, 205). However, in 1945 the seminal
sugarwaspurified and’identified by Mann aso(--)fructose (138,141,142).
The chemical identification was based on a) the preparation of the crystalline methylphenylfructosazone, which is one of the few chemicalcompoundsby meansof which fructose
can safely be distinguished from glucose; b) the purification of seminal fructose
until it reached the same specific optical activity as pure crystalline fructose:
[a. 120” = - g2.2O; c) the demonstration that fructose occurs in the semen in free
form and that it accountsfor the whole of the yeast-fermentable carbohydrate which
yields the Seliwanoff reaction; d) proof, obtained through the use of a highly specific
enzyme, glucose oxidase, that in normal semenglucoseis present only in traces or
altogether absent. So far, the following specieshave been examined and found to
contain regularly fructose in semen: man, bull, ram, rabbit, boar, stallion, goat,
guinea pig, rat, mouse,hamster and opossum.The differencesbetween these species
are, however, very considerable.Whereas in bull and goat, for example, the concentration of fructose in semensometimesreachesa level ashigh as moo mg. per IOO ml.,
in boar and stallion it hardly ever exceedsa value of 50 mg. per IOO ml. Human
semen occupies an intermediate position. However, when comparisons are made
between a specieswith fructose-rich semen,such as bull, and onenotoriously poor in
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THADDEUS
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seminal fructose, e.g. boar, it must be rememberedthat the volume of a single boar
ejaculate is almost one hundred times that of bull so that in effect the quantity of
fructose contained in a single ejaculate is about the samein both species.
Source of Fructose. Huggins and Johnson(85) observedin 1933 that the reducing
sugar in human semenis contained in the secretion of the seminalvesiclesand not the
prostate. Similar observations were made shortly afterwards on bull (16), boar (13 2)
and ram (159). When the seminalsugar was at last identified as fructose it became
possibleto survey in a more detailed manner the fructose-generating tissues.It was
found that in some,though by no meansin all mammals,the seminalvesicle functions
as the chief producer of fructose (140, 141, 143). However, even in those speciesin
which most fructose is formedby the seminalvesicles,e.g. bull, ram, boar, an additional
sourcewas located, usually in the ampullar glands (144, 146). In rabbit Davies and
Mann (34) found fructose both in the glundula vesi&aris (a structure corresponding
to seminal vesicles) and in the ampullae, as well as in the prostate gland. In rat
Humphrey and Mann (95) found fructose mainly in the coagulating gland and the
dorsal prostate; it was absent in the seminalvesicle proper and the ventral prostate.
There is no fructose in either the testes or in the epididymal sperm. Thus at the
site of their origin the spermatozoa, still immotile, have no fructose at their disposal.
During their passagethrough the generative tract, however, the spermatozoa come
in contact with the fructose-rich accessorysecretionsat a time when they assumea
high degreeof motility, to maintain which a sourceof energy is required. The energy
is made available through metabolic reactions which constitute the processof fructolysis (141, 145, 146); its final outcome is the formation of lactic acid, which anaerobically accumulates as the final metabolite whereas aerobically it can be oxidized
further by the spermatozoato provide an additional amount of energy (146, 148).
In view of the fact that fructose comesfrom the accessorysecretions and not
from the spermcellsassuch, it is not surprising that in the whole ejaculate there is no
direct relationship between fructose concentration and sperm density. On the contrary, both in man and animals, an inverse ratio is often found between fructose and
sperm concentration in semen.This follows.from the fact that in a particularly dense
sampleof sementhe spaceoccupiedby spermcellswill be larger, and thevolume taken
up by the fluid portion, i.e. the fructose-containing seminalplasma, correspondingly
less.In our experience, someof the highest values for fructose recorded so far were
found in the semenof vasectomized and thus completely azoospermicindividuals.
The actual level of fructose in semendependsclosely upon the secretory ability of
the male accessoryglands, which explains the occurrence of the striking individual
variations in seminal fructose. Obviously the anatomical features of the accessory
glands such as their size, secretory ability and storage capacity are a decisive factor
which determines the output of fructose in the ejaculate. This is an aspect of particular importance in studies on human semenwhere the size and storage capacity
of the seminalvesiclesare subject to exceptionally large individual variations. Harvey
(74) in her recent survey of fructose in 150 specimensof human semenrecorded values
ranging from 5 to 640 mg/Ioo ml.; specimenswith the minimum and maximum
concentration alike came from fertile donors.
Fructose Secretion as an Indicator of Male Sex Hormone Activity. The ‘fructose
Jantiary
IgjI
MALE
ACCESSORY
ORGANS OF REPRODUCTION
35
test’ was originally described by Mann and Parsons (ISI)
and later developed by
Mann, Davies and Humphrey (147) and Lutwak-Mann, Mann and Price (130) ; it is
basedon the finding that testicular hormone activity is reflected in a most sensitive
manner in the capacity of the accessoryorgans to produce fructose and that therefore the actual level of fructose in the accessorygland secretionsprovides an accurate
indicator of endocrine testicular function. In experiments on rats and rabbits it was
shown that seminal fructose disappearsalmost completely within two weeks after
castration and also that the postcastrate fall in the level of fructose can be prevented
or, if already developed, fully restored, by the administration of testosterone.
The test Canbe carried out asa chemical analysis of fructose in the seminalfluid
collected by means of the artificial vagina from an intact animal, or alternatively,
as an assayof fructose content in accessoryorgansof reproduction obtained from the
experimental animal by dissection. The first approach renders possibleobservation
of the sequenceof changesbrought about by castration and hormonal treatment in
one and the sameanimal and eliminates the necessity of sacrificing the animal prior
to analysis. The other procedure is suitable in experiments with animals like rats
where semencollection is difficult. Whichever procedure is chosen,two further modifications are possible. In the so-called ‘maintenance test,’ the hormone treatment
commencesimmediately after gonadectomy; in the other, the ‘regeneration test,’ the
animal is left after castration until seminal fructose completely disappearsand is
then subjected to the hormone treatment. A quantitative assay of the male sex
hormone is best carried out in the form of the ‘regeneration test.’ The procedure in
this caseis briefly asfollows (I 52).
A group of rats castrated at the age of 6 to 8 weeksare left untreated for about 7
weeks; they are then injected in groups of three with different dosesof the material
to be assayedfor its hormonal activity. The injections are continued daily for 2 or 3
weeks.Following the last day of injections the rats are sacrinced,the accessoryglands
dissected,weighed, ground with trichloroacetic acid, and in the centrifuged proteinfree extracts an assay is carried out of either fructose alone or, better still, of both
fructose and citric acid; the coagulating glands and dorsal prostate provide the material for fructose analysis; the seminalvesiclesand ventral prostate are usedfor citric
acid estimations. The results for each organ are plotted on the dosage-response
standard curves obtained on castrated rats injected with knows daily dosesof pure
testosteronepropionate ranging from 5 to 100 pg.
The main advantages of the ‘fructose test’ are simplicity and sensitivity; these
qualities make it particularly suitable for the study of early symptoms of endocrine
testicular hypofunction as well as for the detection of minimal androgenic effects.
Examples discussedbelow illustrate the usefulnessof the method.
Time Relationship Between Onset of Secretory Activity in Male Accessoq
Glands and Spermatogenesisin Testis. In the courseof investigations by Davies and
Mann (34) on the development of the reproductive functions in rabbit, it was noticed
that fructose appeared in the accessoryglands at an early age when there was as
yet no sign of active spermatogenesis;in the particular breed of rabbits both the gl.
vesicularis and the prostate showedalready at 4 months a fairly high concentration
of fructose in spite of the complete absenceof spermatozoa in the testis or the epi-
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THADDEUS
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didymis. When the spermatozoa appeared at last during the 6th month of life, the
accessory glands were already filled with fluid containing a high level of fructose. In
a similar study of bull-calves Mann, Davies and Humphrey (147) found that fructose
accumulated in the vesicular secretion at the age of approximately 4 months whereas
the first mature spermatozoa appeared nearly 8 months later, Thus, it seems to be the
function of the male accessory glands at that early period of life to prepare a store of
nutrient material in the form of fructose, so that when the spermatozoa begin to pass
through the generative tract they can avail themselves of the fructose reserve. Since
the secretion of fructose depends entirely on the male sex hormone, it must be concluded that the testicular hormone begins to function in the male body well in advance
of the actual spermatogenesis. The possibility cannot, of course, be excluded that
the male sex hormone may be active even at a stage preceding the onset of fructose
formation in accessory organs, but if so, then either its concentration is too small to
produce a marked response in the accessory organs or else its action is countered in
some unknown manner by other factors which operate at the very early age in the
male body.
Early Effects of Testosterone on the Appearance of Fructose in Castrated
Animals. The following experiment has been carried out by Mann, Davies and
Humphrey (147).
Six bull-calves were castrated when I to 2 weeks old, i.e. at an age
prior to the appearance of fructose in the seminal glands. Seven months later two of
the castrated calves were implanted subcutaneously with 0.5 gm. pellets of pure
testosterone, whereas the remaining four were left untreated. After another four weeks
all six animals were slaughtered.and their seminal glands dissected out, weighed and
analyzed both chemically and histologically. The unused portions of the hormone
pellets were recovered from the subcutaneous tissue of the two hormone-treated
calves; their weights were 0.344 and 0.338 gm. respectively, showing that the
quantities of testosterone absorbed per month per animal were 0.156 and 0.162
respectively. The chemical analysis revealed a high content of fructose in the seminal
glands in response to the four weeks’ treatment (51 mg. fructose/Ioo
gm. tissue or
5.3 mg. fructose/total
gland), as against a negligible fructose level in the untreated
castrates (8 mg/Ioo gm. or 0.25 mg/total gland). However, in comparison with and
in contrast to the conspicuous chemical difference, the evidence for the functional
recovery in the seminal glands, as assessed by the histological examination, was
practically imperceptible.
A further illustration of the application of the fructose test to demonstrate an
early effect of androgen on the secretory function of accessory organs was provided
recently by Rudolph and Samuels (196) who found a significant increase in the
fructose content of rat accessory organs IO hours after the injection of one mg.
testosterone propionate.
Assay of the Androgenic Potency of Progesterone by Means of the Fructose
Test. To determine the androgenic effect of pure progesterone, Price, Mann and
Lutwak-Mann
(184) used castrated male rats some of which were injected with
progesteroneand some,for comparison, with small dosesof testosteronepropionate.
3fter a 3lweek period of injections, the animals were autopsied and their accessory
glands analyzed; the values for fructose and citric acid indicated that large dosesof
January
rggr
MALE
ACCESSORY
ORGANS
OF
REPRODUCTION
37
progesterone are capable of producing androgenic effects. It was found that the
androgenic value of 25 mg. pure progesterone is just above that of 0.005 mg. testosterone propionate. This indicates that the corpus luteum hormone has a distinct
androgenic activity which we assessed at not less than 1/5000,
but not more than
1/2500,
of the corresponding dose of testosterone prioponate.
Mechanism of Fructose Formation. Whereas the presence of fructose as a normal
constituent of seminal plasma is a well established fact, information is still lacking as
to how male accessory glands generate this rather unusual sugar. There is no doubt
that in an as yet obscure way the testicular hormone must be involved, directly or
indirectly, in the metabolic formation of fructose. The evidence available at present
points strongly towards the existence in the accessory gland tissue of a metabolic
mechanism whereby blood glucose is converted to seminal fructose, with glycogen
and phosphohexoses functioning as intermediary compounds. Already in 1948 Mann
and Lutwak-Mann
showed (148) that small amounts of fructose can be obtained by
incubating irz vitro minced bull seminal glands with glucose. Subsequentinvestigations on whole animalsby Mann and Parsons(I 52) revealed the existenceof a definite
relationship between the level of glucosein blood and that of fructose in semen.The
link-up between blood glucose and seminal fructose has been studied in rabbits
rendered diabetic by meansof alloxan. In experimental diabetes a five-fold increase
in the blood glucose level led to an approximately equal rise in seminal fructose,
whereas a fall in blood glucose due to insulin invariably produced a considerable
reduction in the fructose of semen,followed by a return to the high level when the
effect of insulin wore off. In connection with these studies one may add that conditions similar to those found in diabetic rabbits were also found in men suffering from
diabetes mellitus; human diabetic semenwas found by Mann and Parsons (152) to
have a fructose content distinctly above the normal range. It is interesting to recall
that already someyears ago Goldblatt (59) noticed an abnormally high reducing
sugar value in human diabetic semen but attributed this mistakenly to urinary
glucose.
The findings on the relationship between blood glucoseand seminalfructose led
to the problem of the mechanismconcerning the enzymic conversion of glucoseinto
fructose. It has been known for a long time that certain phosphorylated compounds
of fructose, such as 6phosphofructofuranose (Neuberg ester) and I ,&diphosphofructofuranose (Harden-Young ester), are formed as intermediary substancesin the
normal carbohydrate metabolismof muscle,liver, brain, spermatozoaand other animal
organs. Phosphofructose can arise in these tissues either from glucose or from
glycogen. In the case of glucose the process starts with the hexokinase-catalyzed
reaction between glucose and adenosinetriphosphate which leads directly to the
formation of 6-phosphoglucose(Robison ester), and then, with the participation of
phosphohexose isomerase, to the production of 6phosphofructose. Concerning
glycogen, the metabolism is initiated by the phosphorylase-catalyzed reaction between glycogen and inorganic phosphate resulting in the formation of r-phosphoglucose(Cori ester) which is first converted by phosphoglucomutaseinto 6-phosphoglucose, and then by phosphohexoseisomeraseinto 6-phosphofructose.In most
animal cells, including mammalian spermatozoa (139, 141, 146), 6-phosphofructose
38
THADDEUS
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LUTWAK-MANN
Volwne
31
is phosphorylated further by adenosinetriphosphate
to I : 6-diphosphofructose
and
fmally converted to lactic acid. However, our recent experiments with male accessory
glands have shown that these tissues possess enzymes which dephosphorylate both
6-phosphofructose and I :6-diphosphofructose
to inorganic phosphate and free sugar;
these experiments were carried out with tissue slices, pulp and extracts from accessory organs of several species but mainly with the seminal vesicles of bull (IL&.
The seminal vesicles in bull (known also as seminal glands or vesicular glands)
consist of two elongated organs of the lobulated tubulo-alveolar type, about IO cm.
long and 3 cm. wide; each lobule is supplied with a duct which branches off the main
collecting duct. By pressure on the gland a clear yellowish-coloured
fluid secretion
can be obtained directly from the main collecting duct; This secretion is very rich in
fructose (17~ or more) but has only a trace of glucose, and contains neither glycogen
nor phosphofructose. The glandular tissue itself, on the other hand, has less fructose
than the secretory fluid but is distinguished by a high content of glycogen (up to 1%
or even more) and a low content of phosphofructose (about 0.027~);
in the fresh state
the gland contains no more than a trace of glucose.
We found in bull seminal glands both amylolytic and phosphorolytic enzymes. On
incubation of freshly minced glandular tissue glycogen disappears and glucose (estimated by means of glucose oxidase) is quickly formed. At the same time, however,
the glands also possess a phosphorylating mechanism which can best be studied with
extracts from minced vesicles subjected to a short dialysis. When r-phosphoglucose is
incubated with such extracts, a mixture is formed consisting of 6=phosphoglucose,
6-phosphofructose, free glucose and free fructose. The proportion of these products
varies according to experimental conditions. At PH 7 the accumulating products
consist in a larger measure of 6-phosphoglucose and 6-phosphofructose;
at higher pH
there is a larger proportion of free glucose and free fructose.
The 6-phosphoglucose and 6-phosphofructose incubated with glandular extract
by themselves are rapidly converted into an equilibrium mixture of both 6-phosphohexoses, and on further incubation they yield a mixture of free glucose and free
fructose. The sugar-liberating phosphatase is activated by Mg ions, and its optimal PII
is about 9.3, corresponding to that of the so-called alkaline phosphatase. However,
the activity, though less pronounced, is quite appreciable already at PH 7 to 8. Both
phosphohexose isomerase and phosphatase are also present in the seminal plasma.
Ram seminal plasma was found to be particularly active toward monophosphohexoses, and also towards I : 6-diphosphofructose from which it liberates both phosphate groups. After one hour’s incubation at 37°C. in presence of 0.005 M-MgClz,
5 mg. substrate (Na salt) acted upon by one ml. dialyzed ram seminal plasma at pH
7 or by 0.2 ml. dialyzed ram seminal plasma at PH 9, yielded the following:
Fructose
PH=7
r -Phosphoglucose
. ... .. ... ... ... ... .. ... ... ... I
6-Phosphoglucose .
............................
r7
6-Phosphofructose
...........................
.23
6-Phosphomannose
..........................
.23
x:6-Diphosphofructose .
.......................
7
I-Phosphofructose ...........................
.6o
(%)
INORG.
77
57
92
79
67
67
94
97
93
IO0
roe
43
49
94
100
5
15
20
95
0
4
60
IOO
PH=7
21
30
I5
70
06
PHOSl'HATE
(%I
GLUCOSE
PH=9
I
65
pH=9
Pa=7
PH=9
hflZW~
IJl5I
MALE
ACCESSORY
ORGANS OF REPRODUCTION
39
The fact that out of a mixture of glucoseand fructose, only one sugar, fructose,
is found in the secretionof the male accessoryglandsmay be due to the reutilization
of glucoseby the glandular tissueitself. In this connection our early observation may
be recalled that slicesfrom the rat accessory tissue can glycolyze glucosebut not
fructose (148). In rat, the sugar-liberating phosphataseis present not only in the
coagulating gland but in other parts of the accessorysystem as well. The concentration of the enzyme decreaseson castration but can be restored with testosterone
propionate. Both in vivo and in vitro experiments suggestthat the following enzymic
reactions may be involved in the formation of seminalfructose (149) :
Glucose (blood) + Glycogen + I-Phosphoglucose + 6-Phosphoglucose + Glucose (reutilized)
6-Phosphofructose + Fructose (secreted)
The finer mechanism of this dephosphorylation process, and its existence in
other animal organs remain to be explored. It is noteworthy that another fructoseproducing tissue,the mammalian placenta, hasbeen known for a long time to be one
of the richest sourcesof alkaline phosphatase (188) directed among others, against
diphosphofructose (40).
CITRIC ACID IN MALE
ACCESSORY ORGANS
Citric Acid in Semen. Citric acid is not a general constituent of animal tissues
and body fluids; like fructose, the occurrenceof which is limited to the male accessory
glands, semen,placenta and foetal fluids, citric acid occurs in relatively few tissuses
and fluids, bones,mammary gland, milk, thyroid gland, urine, male accessoryorgans
and semenbeing the main sources.Most higher mammals including man, bull, ram,
boar, stallion, goat, rat, rabbit and guinea pig possessa fairly high concentration of
citric acid in semen(6, 7, 95, 96, 201, 202). Humphrey and Mann (95, 96), who made
a survey of the citric acid distribution in several species,found the highest concentration, up to one per cent, in bull semen,followed by ram (0.11-0.26%),
rabbit
(o.II-o.&),
boar (0.13%) and stallion (0.06%). With the exception of the rabbit,
citric acid is usually absent from the epididymis and epididymal sperm. It is present,
however, in the ampullae (bull, ram) although in a much smaller quantity than in
ejaculated semen.
From the methodologicalpoint of view it is interesting to note that with fructose
and citric acid alike the clue to the discovery in semenwasprovided by the application
of enzymes as tools for chemical identification. The first observation concerning the
occurrence of citric acid in semenwas made through the useof Thunberg’s methylene
blue technique and citricodehydrogenase,prepared from cucumber seeds(201).
The
use of another enzyme, glucose oxidase from a mould, AspergiZhs niger (135, 136),
made it possibleto exclude the presenceof glucosein semen,and thus helped directly
in the discovery of fructose in semen.
The Formation of Citric Abid and of Fructose as Two Independent Processes.
Although both citric acid and fructose are generated in the same part of the reproductive system, that is, in the accessoryglands, their formation often takes place
40
THADDEUS
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Voltime
31
in anatomically distinct tissues. In rabbit, citric acid is produced chiefly in the
glandula vesicularis, whereas the highest concentration of fructose occurs in the
prostate. In rat, a high concentration of citric acid is present in the seminal vesicle
and ventral prostate, whereas fructose is formed in the coagulating gland and dorsal
prostate. In man, the prostatic secretion, which is virtually devoid of fructose, is at
the same time rich in citric acid. On the other hand, in bull, ram, boar, stallion and
guinea pig, citric acid and fructose are found together in the vesicular secretion. There
are indications that the two substances may be secreted independently by different
cells. A study by Mann, Davies and Humphrey (147) of the bull seminal vesicle has
shown that the secretory epithelium is composed of three distinct types of cells,
designated A, B and C, which appear to be concerned in the secretory processes, but
react in a different manner to several histological stains. In tissue fixed with osmic
acid solution the basally placed cells I? are found to be filled with a lipid staining
characteristically black and forming an irregular ring of large droplets around the
tubule. Neither A nor C cells contain the lipid, but again the two types of cells differ
from each other, in so far as staining is concerned. It remains, however, for further
study to establish which of them is specifically concerned with the production of
either citric acid or fructose.
Dependence on the Male Sex Hormone. Following castration, citric acid gradually disappears from the accessory gland secretions but on implantation or injection
of testosterone it reappears again (95, 96, 147, 150).
A direct relationship exists
between the amount of testosterone injected into castrated rats and the response of
the rat seminal vesicle to produce citric acid. This makes it possible to use the quantitative assay of citric acid in the same manner as that of fructose, as a sensitive and
simple her-mone indicator test (152).
Hypophysectomy
produces an effect similar to
castration, and thesecretionof citric acid by the gl. vesicularis of a hypophysectomized
rabbit can be restored either by testosterone or by gonadotrophin (152).
Secretion of Citric Acid in the Female Prostate. A glandular structure corresponding to the male prostate gland develops occasionally in the female body. It has
been described in the human species but most studies concerning the female prostate
have been done with rats (21, 37, 71, 109, 134, 181, 182). In rat this organ is located
in a position corresponding to that of the male ventral prostate which it also resembles histologically. It reacts to stimulation by androgens and is inhibited by
estrogens. The incidence of the female prostate in rat is usually no more than a few
per cent; as a rule the organ attains a certain degree of histological development during
the first few weeks of life, during the last part of pregnancy, and during the first 2
weeks of lactation; otherwise it remains completely regressed and inactive.
By inbreeding it is possible to raise considerably the incidence of the female
prostate in rats. Using such an inbred stock, Price, Mann and Lutwak-Mann
(184)
were able to show that the analogy between the female prostate and the male ventral
prostate extends to the chemical character of the secretion and that the female
counterpart of the male ventral prostate also produces citric acid. In a state of
quiescence the average weight of female prostate in a a-month-old rat was 4 mg.
and the citric acid content 2 p-c&.However, in response to stimulation with testosterone propionate (daily injections of 200 pg. for 3 weeks) the average weight of the
female prostate rose to I I 2 mg. and the citric acid content to 125 pg. In male rats of
January Igp
MALE
ACCESSORY
ORGANS
OF
REPRODUCTION
41
comparable age the citric acid content of the ventral prostate was I 21 pg. per organ.
The female prostate, like the male ventral prostate, doesnot secretefructose.
Function of Citric Acid. In sofar as its nutrient role is concernedcitric acid would
seemto be of much lessvalue to spermatozoathan fructose (gs,g6). It hasbeenshown,
however, to have some beneficial influence on the sperm motility (116). It is also
conceivable that it may be connected with such phenomena as coagulation and
liquefaction of semen (87) or the calcium-binding capacity of seminal plasma (80).
Another possibility is a link with the hyaluronidase activity as indicated by Baumberger and Fried (3) who found that citrate exerts a ‘protective action’ against
antinvasin iuzvitro. It is interesting to note that in rabbit, where the phenomenonof
semengelation is particularly well developed, citric acid is associatedchiefly with
the gel formation in the gl. vesicularis, and not with the fluid secretionof the prostate
(96, 152). Similar conditions are present in the rat. The seminalvesicle, which is the
chief site of citric acid in rat, is also remarkable for its very low aconitase content
(96). One is almost inclined to believe that there may be a connection between the
low level of aconitase and the high level of citric acid, and that, perhaps, citric acid
accumulatesbecauseits further breakdown is prevented by the absenceof aconitase,
It is interesting to recall here the results of the recent studies on the ‘citric acid condensing enzyme’ of liver tissue which indicate that aconitase is essentialfor further
breakdown of citric acid (210). Another fact which may also bear some relation to
the mechanismof citric acid accumulation in the seminalvesicle concernsthe presence
in the seminal plasma of a heat-labile factor which inhibits the enzymic breakdown
of citrate by liver tissue (96). On the other hand, it should be mentioned that the
human prostate, another citric acid producing organ, has been stated to possessa
high content of aconitase (6).
HORMONE-INDUCED
SECRETORY PROCESSES IN TRANSPLANTS
ACCESSORY GLANDS OF REPRODUCTION
FROM MALE
It has been shown in the preceding section that the secretory activity of the
accessoryorganscan be followed and quantitatively assessed
by meansof the chemical
hormone-indicator tests. It was of interest to determine to what extent the function
of the accessory glands depends upon the preservation of intact anatomical and
neural links with the rest of the male sex apparatus. Insight into this problem was
gained through the technique of subcutaneoustransplantation of the organs and a
study of the behavior of these grafts under various experimental conditions. In this
manner it wasdemonstrated (130, ISO, 184) that transplanted accessorygland tissues
are capable of producing and accumulating considerablequantities of fructose and
citric acid, in complete anatomical separation from the male reproductive tract,
provided, however, that they remain under a constant influence of the male sex
hormone.
Tissue fragments not exceeding one mg. fresh weight were dissectedfrom the
rat seminalvesicle and coagulating gland of 40- to so-day-old male donors and implanted subcutaneously into 40. to Iso-day-old male hosts. After 3 months’ subcutaneous development, when the grafts could easily be palpated through the skin,
they were dissectedand analyzed. Those from the coagulating gland alone contained
only fructose, but in those obtained from the combined coagulating gland and seminal
42
THADDEUS
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CECILIA
LUTWAH-MANN
Volume
31
vesicle tissuethere were both fructose and citric acid. This showsthat the tiny fragments of young tissue from which the grafts grew were endowed with the samebiochemical potential as the correspondingglands in the intact body. In fact, on several
occasionsit was noted that the transplants had actually a higher fructose and citric
acid content than the corresponding intact glands of the graft-bearing male hosts.
This is presumably due to the fact that, unlike the intact seminal vesicle or coagulating gland, a graft has no outlet, Incidentally, this observation provides an
illustration to the finding previously obtained in in vitro experiments, that once
produced by the secretory epithelium, fructose or citric acid do not readily undergo
reabsorption, at least not for so long as the tissue continues active secretion. However, when graft-carrying male rats were castrated the result was a considerablefall
in the fructose and citric acid content not only in the host, glands but in the grafted
tissuesas well. The postcastrate fall could, however, be prevented by administration
of the male sexhormone. Coagulating gland transplants in castrated male rats treated
for several weekswith a daily doseof 200 pg. testosteronepropionate showed144 mg.
per cent fructose; however, interruption of hormone treatment for three weekscaused
a decline in the grafts to 12 mg. per cent.
The behavior of grafts of male glands in female rats furnished further convincing
proof of the close relationship between the function of testicular hormone and the
metabolic activity of coagulating gland tissue, In a seriesof experiments (130) spayed
and non-spayed female rats were used as hosts, Successfuldevelopment of male
accessory tissue transplanted under the skin of female hosts was achieved in all
instanceswhere the animals were kept under continuous treatment with testosterone
propionate. After a period of three to eight weeks’ administration of a daily doseof
200 pg. hormonethe grafts removed from the subcutaneoussites showedas much as
200 mg. per cent fructose. In agreementwith the results gained with male hosts, the
cessationof hormonal treatment causeda decline in the fructose-forming activity of
the grafts in the female hosts. In all these experiments there was no evidence that
ovaries interfered with graft development, sinceresults were identical in normal and
spayed rats.
In another study Price, Mann and Lutwak-Mann (184) demonstrated that transplants of rat accessory organs (coagulating gland and ventral, dorsal, and lateral
prostate) can be successfullygrown in female rats which following the subcutaneous
implantation, were injected with equine gonadotrophin, 20 international units of
purified pregnant mare serumgonadotrophin, daily for 30 days. At autopsy the ovaries
of the female hostswere found to be at least ten times enlarged, and the grafts had a
high content of fructose and citric acid, of the sameorder as in experiments with
testosterone propionate. Again, the coagulating gland transplants contained only
fructose and no citric acid, whereasthe ventral prostate grafts had citric acid but no
fructose. In these experiments gonadotrophin stimulated vigorously
the hormonal
production of the ovaries and the increasedoutput of ovarian androgens
was sufficient
to induce secretory processesin transplants from male accessorygland tissue.
SECRETION OF PHOSPHATASES
The mammalianseminalplasmacarriesa variety of highly active enzymes which
originate in the accessoryglandsand are essentialcomponentsof the secretory fluids.
Jmwlr
y I#I
MALE
ACCESSORY
ORGANS
OF
REPRODUCTION
43
So far, however, few of these enzymes have been purified to any appreciable extent,
mainly because it is difficult to secure sufficient quantities of material. This explains
to some extent the confusion which exists with regard to the identity, range of activity and nomenclature of most of these enzymes. The position of the so-called
‘acid phosphatase’ and ‘alkaline phosphatase’ illustrates well the present situation in
this respect. Both terms derive from the fact that most, though by no means all,
substrates attacked by the seminal plasma are dephosphorylated best at either an
acid pH, usually about 5, or at alkaline PH, usually about g. This however, does not
mean that the ‘acid’ and ‘alkaline’ enzyme are the only two, or even the principal
two, phosphatases of the accessory secretions. Some time ago Reis discovered in
human seminal plasma the powerful 5-nucleotidase, an enzyme specifically concerned
with 5-nucleotides such as adenylic acid (188-191).
The situation is further aggravated by the fact that some investigators continue to use whole semen as the
source of phosphatases, without drawing a distinction between the enzymes of the
seminal plasma and those of the spermatozoa. Yet, as pointed out before (146), this
is essential, particularly in the case of enzymes concerned with phosphorus metabolism of semen, which are distinctly different in sperm and plasma.
In most earlier studies on phosphatases, phosphoglycerol (66, IIS) and also
6-phosphohexose (192) were used as substrates, but more recently other phosphoric
acid esters have been employed as well as certain synthetic derivatives particularly
suitable for quantitative assays of phosphatase activity; two such substrates now
widely used are phenylphosphate introduced by King and Armstrong (107) and
phenolphthalein phosphate, also introduced by King (106).
The latter substance,
itself colorless, yields on enzymic hydrolysis free phenophthalein which turns bright
red on alkalinization and can thus be easily determined calorimetrically (19). The new
procedure of Huggins and Talalay (92) for quantitative assay of both the ‘acid’ and
the ‘alkaline’ phosphatase in semen and in glandular secretions also requires phenolphthalein phosphate as enzyme substrate.
In accessory gland tissue itself the presence and distribution of the phosphatases
can be followed by means of histochemical methods. This procedure originally worked
out by Gomori (61-63) and subsequently developed and applied by several investigators (38, 39, IOI, 157, 158, 206, 223, 224) consists briefly in incubation of
tissue sections in solutions of organic phosphates suitably buffered, the precipitation
of the liberated phosphoric acid in the form of lead or calcium salt, and the staining
of the precipitate by conversion into cobaltous sulfide or by impregnation with
silver or alizarin.
Acid Phosphatase.
An observation that the phosphatase activity of urine is
usually higher in men than in women prompted Kutscher and Wolbergs (I 13) to
examine the phosphataseactivity of human semen.This led to the discovery that
semenis one of the richest sourcesof phosphatasein the human body, and that the
enzyme has its origin in the prostate gland. The prostatic enzyme has been classified
as an ‘acid’ phosphatasewith an optimum activity at acid PH towards both cy-and
P-phosphoglycerol, but largely inactive towards diphosphofructose and pyrophosphate (I I 2). Subsequentinvestigations confirmed and extended thesefindings. It was
shown by Scott and Huggins (204) that while the voided urine of man is rich in
‘acid.’ phosphatase, urine obtained directly from the renal pelvis by catheter con-
44
THADDEUS
MANN
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CECILIA
LUTWAK-MANN
Vohte
31
tains only very small amounts of the enzyme. This convinced them that the content
of ‘acid’ phosphatase in male urine is in fact largely due to the admixture of prostatic
fluid and can thus be used as an index of the ‘resting secretion’ of the human prostate.
Prostatic phosphatase like the other constituents of seminal plasma ranks as a
chemical secondary characteristic of the male sex. Investigations by Gutman and
Gutman (69) have shown that the level of the enzyme in the human prostate is low
in infancy, but increases rapidly during puberty; the activity found and expressed
in King-Armstrong
units per gram fresh tissue, was I$ units at 4. years of age, 73
units at puberty, and 522 to 2284 units in adult men. A similar relation to age was
observed in monkey (70) and dog (88); in both these species injections of androgenic
hormones to immature males stimulated considerably the output of ‘acid’ phosphatase
from the prostate. However, like with other constituents of semen, the level of the
prostatic phosphatase activity varies from one species to another as well as within
the same species (so). Canine prostate, for example ? does not exceed 70 units of
phosphatase per gram tissue, as compared with 4700 units recorded in monkeys
(81);
in rat (94) and rabbit (13, 69) the level of ‘acid’ phosphatase is even lower than
in dog. In man the ‘acid’ phosphatase does not normally pass from the prostate into
the blood stream. However, it is found in blood as a result of malignant growth in
the prostate with metastases; this has been utilized as an important clinical aid for
the diagnosis of cancer, and as a follow-up technique during treatment, indicative
of the involution of metastases (84? 90, 91).
Choline and Phosphorylcholine.
Relation to Acid Phosphatase. Mammalian
seminal plasma is distinguished by a high content of choline. The concentration in
*human semen is so high that the presence of choline was utilized in forensic medicine
for the purpose of semen detection; the old-established ‘Florence test’ (56) for sperm
stains depends on the formation of characteristic crystals with potassium triiodide.
Following an observation that the content of choline is very low in freshly ejaculated
semen (IO mg.%) but increases steadily on storage (860 rng.70 after IO minutes,
1600 mg.% after I hour, 2120 mg.% after 6 hours) Kahane and Levy (102) have
shown that fresh semen contains a ‘precurseur de la choline’ from which free choline
can be liberated enzymically either by dilute semen solutions or by prostate extracts.
They established the presence of the choline precursor in most reproductive organs
including testes of bull, boar, ram, stallion, rabbit and guinea pig; seminal vesicles
of stallion and guinea pig, and epididymis of boar and ram, but found none in the
prostate of dog, stallion and ram. Later researches (103, 104) led to the conclusion
that the precursor is probably choline glycerophosphate. It was also found that the
quantity of choline set free enzymically from the precursor is far in excess of the
simultaneously appearing inorganic phosphate, and that a major part of choline
must therefore arise through the action of yet another enzymic process.
The nature of the phosphorus compounds in freshly ejaculated human semen,
from which inorganic phosphate is liberated on incubation, was investigated by
Lundquist (I 24-126). He isolated from fresh human semen phosphorylcholine and
showed that a few minutes after ejaculation of semen this substance undergoes
rapid enzymic decomposition to free choline and inorganic phosphate; there was
21.6 mg. per cent inorganic P in human semen frozen to- IoOC. immediately after
Jamary
xgp
MALE
ACCESSORY
okaANS
OF REPRODUCTION
45
ejaculation, but 64.2 mg. per cent after 20 minutes, and 64.5 mg. per cent after
34 minutes, incubation at + 2o°C. According to Lundqukt
the enzyme
responsible
for the dephosphorylation of phosphorylcholine in human seminal plasma
is the
‘acid’ phosphataseof the prostatic secretion, and the optimal pi of hydrolysis
is
6.3 (in acetate buffer). But the presenceof yet another phosphatase
in human
semen
active towards phosphorylcholine at a higher pH has been describedby Hudson and
Butler (78). The distribution of phosphorylcholine in the male reproductive
organs
has not been investigated, but it has been pointed out in 1933 by Huggins
and
Johnson (85) that the major part of the phosphorus content of human seminal
plasma comesfrom the seminalvesicles.From this Lundquist infers that phosphorylcholine is formed in the seminalvesicles, and that the dephosphorylation is due to
contact establishedon ejaculation, between the vesicular secretion which provides
the substrate, and the prostate secretion which contains the phosphatase.
Alkaline Phosphatase.This enzyme is characteristic of the male accessory
organs
as a whole but its distribution doesnot coincide with that of the acid phosphatase.
Human semen,with its notoriously high level of the prostatic or ‘acid’ phosphatase,
has a low concentration of the ‘alkaline’ enzyme. Bull semenhas a very low level of
the ‘acid’ enzyme, but a slightly higher content of the alkaline phosphatase (72,
187). The difference, however, between the human and bovine semenis not altogether
unexpected if one recalls that the bulk of seminalplasma in bull is derived from the
seminalvesiclesand not from the prostate. On the whole, the prostatic secretion as
such is but a poor source of the alkaline phosphatase.This however should not be
mistaken for an absenceof alkaline phosphatasein the prostate gland itself. As a
matter of fact, the example of prostate showshow essentialit is to distinguish between
secreted and non-secreted enzymes. Using the histochemical method Gomori (62)
was able to show that the human prostate contains both the acid and the alkaline
phosphatase.Whereas, however, the ‘acid’ enzyme occurs in the epithelial secretory
cells themselves,the other phosphataseis confined largely to the walls of the blood
capillaries.
In rat both phosphatasesoccur in the seminalvesicle, coagulating gland, ventral
prostate and dorsolateral prostate. With the exception of the ventral prostate which
may contain up to 20 units of alkaline phosphataseper gram tissue, the level of
either enzyme seldomexceeds4 units per gram in any of the organs (94, 208). After
castration both enzymes undergo reduction in activity, first in the seminalvesicles,
and a few days later in the prostate, but the percentage decreaseof activity is about
the sameas that of organ weight. The activity of both enzymes can be restored by
testosteroneroughly to the sameextent (208). But in spite of this apparently similar
behavior these two enzymes contribute to a varying degreeto the secretory function
of the individual organs. The rat seminalvesicle examined histochemically exhibits
only a very faint reaction of alkaline phosphatasein the secretory cells themselves,
but beneath the epithelium, in the reticulum and endothelium of the stroma and
basal membrane, the reaction is very strong indeed (38). A similar picture has been
observed in the mouse seminal vesicle (2). Clearly, the alkaline phosphatasesof
the rat and mouse seminal vesicle belong to the category of ‘stromal’ rather than
‘epithelial’ or ‘secretory’ enzymes. Equally interesting is the pattern of phosphatase
46
THADDEUS
MANN
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LUTWAK-MANN
Volzcme 31
distribution in the remaining rat accessory organs. According to Bern (14) who recently investigated 7 species (rat, mouse, hamster, guinea pig, rabbit, bat and
opossum),the rat ventral prostate, unlike the seminal vesicle, contains the alkaline
phosphatasemainly in the secretory cells and in the secretion itself, but not in the
stroma. On the other hand the coagulating gland and the dorsal prostate do not
secretealkaline phosphataseand the enzyme is present only in the stroma.
Relation Between Fructose Formation and PhosphataseActivity. The acid as
well as the alkaline phosphatasehave both been credited in recent years with participation in the processof fructose formation (14, 78, 206). With regard to the acid
(prostatic) phosphatase the evidence has been unsatisfactory. The rates at which
6-phosphofructose and I : 6-diphosphofructose are dephosphorylated by the ‘acid’
enzyme, are on the whole rather small. The suggestionthat in analogy to phosphorylcholine, fructose may be secreted as phosphofructose and dephosphorylated after
ejaculation of semen, by the prostatic phosphatase, lacks sufficient experimental
evidence. On the contrary, our researcheson many mammalian specieshave proved
that the bulk of fructose is secretedin a free, non-phosphorylated and yeast-fermentable form. Bull seminal plasma and vesicular secretion are the richest source of
fructose in the animal body but at the same time they are conspicuously poor in
inorganic phosphate. One would expect that if the high concentration of fructose
(IQ/O or more) in bull seminalplasma were the end product of a processanalogous
to the breakdown of phosphorylcholine in semen, then the content of inorganic
phosphate in bull seminalplasmashould be IO to 20 times higher than it is. Similarly,
all claims regarding the presence of substantial quantities of phosphofructose in
accessorygland secretionsshould be treated with reserve unlesssupported by chemical isolation and proper identification.
Alkaline phosphataseis capable of hydrolyzing phosphorylated fructose at a
considerablerate. As already pointed out in the section on Mechanism of Fructose
Formation, it may well play a role in fructose formation, There is no experimental
evidence as yet that phosphofructose is secreted as such by one gland and then
dephosphorylated by an enzyme secreted by another gland. What evidence there is
at present points to the existence of an intracellular enzymic system which acts
upon blood glucoseas the initial substrate, and leads through glycogen, r-phosphoglucose, 6-phosphoglucose,6=phosphofructose,and possibly, I : 6-diphosphofructose,
to free fructose (I&.
When the formation of free fructose has been accomplished
in the secretory epithelium, it then passesinto the lumen of the accessorygland and
with the fluid secretion into the urethra. Since differences have been demonstrated
in cellular distribution between the ‘secretory’ and ‘stromal’ alkaline phosphatase,it
is conceivable that the formation of fructose may be related to one and not to the
other type of enzyme. Moreover, the possibility cannot as yet be excluded that there
is an enzymic entity distinct from both types of the ‘alkaline’
enzyme,
concerned
specifically with fructose-phosphates.
S-Nucleotidase. Reis (W-rgo)
was first to describe this enzyme which
dephosphorylates muscle adenylic acid (adenosine-5’-phosphoricacid) and inosinic
acid but is inactive towards adenosine-triphosphateand yeast adenine nucleotide
(adenosine-3-phosphoric acid). The mammalian seminal plasma, especially in bull,
January rgjI
MALE
ACCESSORY
ORGANS
OF
REPRODUCTION
47
is a particularly rich source of the enzyme; the rate at which adenylic acid is dephosphorylated by the bull vesicular secretion exceeds several hundred times that of
,&phosphoglycerol (137, 143).
ROLE
OF ACCESSORY
GLAND SECRETIONS
IN THE
COAGULATION
AND
LIQUEFACTION
OF SEMEN
Bouchon Vaginal. Mammalian semenis ejaculated in a liquid form. In some
animalssuch as bull and dog, it remainsliquid, while in others it undergoes
undergoesrapidly
rapidly a
a
processof peculiar gelation or coagulation. The phenomenonof coagulation is particularly striking in rodents where it leads to the formation of the so-calledbo&on
bowlion
vugind or vaginal plug, after mating. It is generally believed that the purpose of
vaginaI
the copulatory plug is to prevent the outflow of semenfrom the vagina,
vagina. The plug
probably also assiststhe passageof spermatozoa through the cervix into the uterus;
in a recent study of sperm transport in the rat it has been shown that if the coagulation of semenis prevented by ligation of the ducts belonging to the seminal vesicles
and the coagulating gland, the ejaculate doesnot passthrough the cervix uteri (18).
(18).
In addition to rodents the occurrence of the copulatory plug has been recorded in
Insectivora (moleand hedgehog),Chiroptera (Rhinolophidaeaswell asVesfertilionidae)
and Marsz#dia
Marsz@alia (24, 32, 43-45, 52, 194, 212). Whereas however, in most of these
animals the vaginal plug is the result of the coagulation of semenitself, in some,
namely in the opossum(73) and in the bat Vespemgano&da (31, 65), its formation
is more complicated and involves the coagulation of female secretory products by
the seminal plasma, and in others it is made up from the vaginal epithelium.
To a certain extent the phenomenonof semenclotting occursalsoin higher domestic
animals such as boar and stallion, and in the primates. In boar a semenejaculate
inspected shortly after delivery from the urethra contains only small lumps of
tapioca-like gelatinous material which soon increasein size and finally merge into
one solid massof gel. Human semenforms a clot immediately after emissionbut it
usually liquefies spontaneously after an interval of 15 minutes. The coagulation
phenomenon occurs also in monkeys.
Vesiculase. Already in the last century, mainly through studies on rodents,
several aspectsof the coagulation phenomenonwere known, such asfor instance that
it is essentially a property of the seminal plasma, and probably due to interaction
between a protein-like coagulable substrate secreted by the seminal vesicles and a
catalytic agent which is not present in the vesiclesthemselvesbut comesinto contact
with the coagulable protein in the course of ejaculation (II, 17, 76, IIS, 117, 178).
The first to recognize the enzymic nature of the coagulating catalyst were Camusand
Gley (23-27). The enzyme was named ‘vesiculase’ and was at first thought to be
formed in the prostate gland; later studies, however, gave more precise information about the distribution of the enzyme. In rat, Walker (219, 220) showedthat the
vesiculaseoriginates in a small gland which he named the ‘coagulating gland’ but
which is sometimesalso referred to as ‘rat anterior prostate’ since morphologically
it forms part of the rat prostate complex (51, 52). The coagulating gland lies closely
adjacent to the seminalvesicle proper with which it is enveloped in a common peritoneal sheath but the two organs open into the urethra through two independent
48
THADDEUS
MANN
AND
CECILIA
LUTWAK-MANN
Volume31
ducts. A similar anatomical separation between vesiculase and the source of the
coagulable material was shown by Walker in the guinea pig.
For the demonstration of the coagulation phenomenon iuz vitro the secretions
are best obtained from rat. For this purpose the coagulating gland and seminal
vesicle are excised with care so as to prevent tissue damage, and the two secretions
are collected separately, diluted with saline and if necessary filtered until clear. As
soon as the solutions are mixed, the coagulum formation sets in until the whole becomes a solid plastic mass. It is possible to show that one part of the coagulating
gland secretion is sufficient to induce the coagulation of more than zo,ooo parts of
the vesicular secretion.
After castration, during the involution period in the accessory organs, the
coagulation phenomenon becomes negative not so much because of the disappearance
of the enzyme, but mainly owing to the failure of the seminal vesicles to elaborate
the protein required as substrate ($3). Moore and Gallagher (164) based on this
fact their ‘coagulation test’ in the guinea pig. The test depends on the induction of
seminal discharge in guinea pig by stimulation with 30 volts alternating electric current applied to the head. Electrically induced ejaculation in normal or castrated but
androgen-treated
animals produces semen which coagulates rapidly. In material
from castrated non-treated animals there is no coagulation. However, the absence
of the coagulation phenomenon in a given ejaculate need not necessarily indicate a
castrate condition in the accessory glands. A normal rabbit, for example, may produce upon occasion as much as 4 ml. semen, the greater part consisting of gelatinous
secretion from the gl. vesicularis, whereas another time the same animal may yield
a small ejaculate entirely devoid of gel. There is also some evidence of species variation as regards the localization of the clotting mechanism. Eadie (45) who investigated the phenomenon in Insectivora came to the conclusion that in Condylura and
Parascalops the coagulable substrate is produced by the prostate gland, and the
coagulating agent by Cowper’s gland. He reproduced successfully the phenomenon
iut vitro by using the fluid from the excised glands of freshly trapped moles. The
mechanism of coagulation in human semen is still uncertain but from experiments
on monkeys it would appear that a substrate-enzyme mechanism may be involved.
In most monkeys the prostate gland consists of two distinct parts; in Maraca mulatta,
van Wagenen (216) found that coagulation is brought about by the action of the
secretion from the cranial lobe of the prostate upon the secretion of the seminal
vesicle; the secretion of the caudal lobe of the prostate is unable to coagulate the
vesicular fluid.
Little is known at present about the nature or specificity of the coagulating
enzyme, except that the enzyme from one species can induce coagulation in the
vesicular fluid of another species. Such cross-coagulation has been demonstrated
between the secretions of rat and guinea pig (220), and between rat and monkey
(216) ; that is to say, the vesicular secretion of monkey can be coagulated by the
coagulating gland enzyme from rat and in turn, the vesicular secretion of the rat
can be coagulated by the enzyme from the cranial lobe of the monkey prostate. So
far. no cross-coagulation could be demonstrated with human accessory secretions,
Fibrinolysin and Fibrinogenase. In human semen coagulation is followed by
January
1951
MALE
ACCESSORY ORGANS OF REPRODUCTION
49
liquefaction which is another enzymic reaction initiated by accessorygland secretions. The proteolytic enzyme concernedwith liquefaction is present in the prostatic
secretion. Its discoverers, Huggins and Neal (87), named it ‘fibrinolysin’ because
of its ability to digest blood fibrin, and its similarity to the fibrinolytic agent discovered earlier in hemolytic streptococci (215).
Since then, however, the streptococcal fibrinolysin has been defined as a kinase, and its function shown to be that
of a substancewhich activates the fibrinolytic enzyme already present in the blood.
Consequently, the name ‘fibrinolysin’ has been abandoned with reference to the
streptococcal agent in favour of ‘streptokinase.’ However, as shown by Oettle (172)
the prostatic fibrinolysin cannot replace streptokinase as activator of the blood
enzyme; nor is it identical with the fibrinolytic enzyme which occurs in the blood
itself. Thus judgement must be reserved at present on the question of the identity
of the prostatic fibrinolysin. In human semen,which is particularly rich in fibrinolysin, the fibrinolytic activity can be conveniently determined by the method of
Harvey (75). This consistsin mixing a constant volume of oxalated blood plasma
with varying volumes of semen,inducing clotting by the addition of calcium chloride,
and estimating the time taken for the clot to liquefy. The individual differences
in the fibrinolytic activity of human semenare considerablebut in any given individual the activity is fairly constant.
Both human and dog prostatic secretioncontain alsoanother proteolytic enzyme,
‘fibrinogenase?’which inactivates fibrinogen (87, 93); the human prostatic secretion
is rich in fibrinolysin but poor in fibrinogenase; the reverse is true of dog.
On incubation of human semenat 37’ C. for a few hours, considerableaccumulation of free amino acids takes place, probably the outcome of the proteolytic action
of the prostatic secretion on a substrate present in the vesicular fluid (128).
Liberation of free ammonia is another processassociatedwith incubation of mammalian
semen,particularly in the ram (137,
139).
NUTRITIONAL
REQUIREMENTSFORTHE
NORMAL FUNCTION
OF MALE ACCESSORY OR GANS
It is well known that starvation or inadequate food intake cause far reaching
changesin the sexual apparatus suchas testicular atrophy, decline of spermatogenesis,
diminished libido, regressionof male accessoryorgans.The changesin the accessory
glands are extensive and often precede other symptoms; on the other hand, these
organs respond promptly and in a characteristic manner to the restoration of adequate food supply. One of the earliest studies on the effect of inanition and malnutrition upon the male reproductive organsin mammalswas carried out by Jackson
(100).
This was followed during the past 25 years by a seriesof important investigations; a thorough discussionof these numerouscontributions is best found in review
articles by Asdell (I), M ason (155>,
Reid (186), Russel (I&,
Samuels (200)
and
Walton (221).
There have been several new contributions to this subject (20, 60,
156, 174, 222). However, the perusal of the literature reveals the need for a drastic
revision of the criteria on which to base the diagnosisof malnutrition effects in the
male reproductive system. Quite often the examination of the spermatozoa and the
spermatogenesishave been relied upon as the sole criterion, without reference to
THADDEUS
50
MANN
AND
CECILIA
LUTWAH-MANN
Volwne
31
the state of the endocrine activity of the testes and the closely related function of
the accessory organs. Yet it is a widely recognized fact that the spermatogenic and
the endocrine activity of the testes are not equally sensitive to the same deficiency;
in male rats, vitamin E deficiency impairs the spermatogenesis, but causes relatively
little damage to the accessories; vitamin B depletion lowers the hormonal activity
and in consequence the secretory function of the accessory glands, leaving spermatogenesis little affected. Another example is the different character of testicular changes
due to vitamin E and vitamin A deficiencies.
Detailed studies were carried out to examine the effect on accessory organs of
general inanition and vitamin B deficiency. In 1931 Moore and Samuels (167) demonstrated that a rapid involution of secondary sex glands followed if male rats were
fed a vitamin B-deficient diet, or if their total food intake was very considerably
reduced. They were able to correct the changes in the accessory glands by means of
testicular hormone injections and also by administration of crude anterior pituitary
hormone. They drew the conclusion that the primary lesion due to inadequate feeding
was located in the pituitary gland. A similar state of ‘pseudo-hypophysectomy’
was demonstrated later in rats (171) and in dogs (177) More recently Lutwak-Mann
and Mann 0
used chemical methods to measure quantitatively
the secretory
function of male accessory glands in normal and nutritionally deficient rats. They
found that a few weeks of vitamin B-deficient diet reduced the content of fructose
and citric acid in the accessory organs to a level almost as low as that of castrated
rats. However, both the secretion of fructose in the coagulating gland as well as the
production of citric acid by the seminal vesicles and ventral prostate could be restored completely by treatment with either testosterone propionate or chorionic
gonadotrophin.
The introduction of chemical methods makes it practicable at last to determine
in a quantitative manner the functional state of the male accessory organs. Equally,
it makes possible the assay of secretory activity of the male accessory gldnds in live
animals, since estimations of fructose and citric acid can be carried out not only in
dissected glands but just as well in ejaculated semen. The chemical approach is
particularly
suitable for investigations concerned with the relationship between
nutrition and reproduction in view of the great sensitivity of the accessory organs
to nutritional deficiencies. Doubtless, other important components of accessory
secretions will be discovered in the future, but even those known at present give
a satisfactory basis for an accurate appraisal of the male sex accessory apparatus.
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