The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 3 997-1001
Copyright © 1999 by The Endocrine Society
Secretion of Testosterone and Its 
4 Precursor Steroids into Spermatic Vein Blood in Men with Varicocele-Associated Infertility1
Stephen J. Winters,
Jun Takahashi2 and
Philip Troen
Department of Medicine, University of Pittsburgh Medical Center,
Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Stephen J. Winters, M.D., Department of Medicine, University of Pittsburgh Medical Center, Montefiore N-919, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. E-mail: winters{at}med1.dept-med.pitt.edu
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Abstract
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Insight into the mechanisms by which steroid hormones are released from
the testes was sought by examining the concentrations of
progesterone, 17
-hydroxyprogesterone, and
androstenedione as well as testosterone in spermatic vein blood every
15 min for 4 h in men with varicocele-associated infertility.
Coincident discrete secretory episodes of all four steroids were found,
and spermatic vein concentrations of testosterone were highly
positively correlated to the concentrations of
progesterone (r = 0.79), 17-hydroxyprogesterone
(r = 0.81), and androstenedione (r = 0.82), respectively. The
sum of the four measured steroids per mL plasma was calculated, and
testosterone was found to account for 70%, 17-hydroxyprogesterone
for 24%, androstenedione for 5%, and progesterone for
1% of the total. In a previous study of the intratesticular steroids
in a separate population of men with varicocele-associated infertility,
the sum of these four steroids per g tissue was similarly calculated.
Testosterone accounted for 70% of the four measured steroids,
17-hydroxyprogesterone for 22%, androstenedione for 4%, and
progesterone for 3% of the total. Thus, the relative
concentrations of these four steroids are nearly identical in
testicular tissue and spermatic vein plasma. From these data we
hypothesize that steroids in the testicular interstitium are cosecreted
into peripheral plasma in response to stimulation by LH and propose
that the mechanism initiating this pulsatile mode of secretion of
testosterone and its precursor steroids may not be coupled to
testosterone biosynthesis.
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Introduction
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THE MECHANISM by which steroid hormones are
synthesized and released from the testis into the circulation is
incompletely understood. Testosterone is synthesized from cholesterol
through a series of enzymatic transformations that occur in the Leydig
cell mitochondria and microsomes (smooth endoplasmic reticulum and
surrounding cytoplasm (1). Cholesterol for steroidogenesis is stored in
ester form in lipid droplets, which are hydrolyzed by LH activation of
cholesterol ester hydrolase. The rate-limiting step in steroidogenesis
is the transfer of cholesterol to the inner mitochondrial membrane for
bioconversion to pregnenolone by the cytochrome P450 side-chain
cleavage enzyme that occurs when LH stimulates the labile regulatory
protein, StAR (2). Pregnenolone is transferred to the smooth
endoplasmic reticulum where it is converted to testosterone via a
series of intermediates that have a double bond in the A ring,
progesterone, 17-hydroxyprogesterone, and
4-androstene-3,17-dione (
4 pathway), or through
intermediates with a double bond in the B ring, pregnenolone,
17-hydroxypregnenolone, dehydroepiandrosterone, and
5-androstene-3ß-diol (5 pathway).
The testicular content of testosterone in adult men is approximately 50
µg/testis (3). Because the daily production rate of testosterone is
5–7 mg in men (4), it is clear that testosterone is continuously
produced and released into the circulation. It is generally believed
that testosterone produced by Leydig cells diffuses into interstitial
fluid and then enters testicular capillaries or enters capillaries
directly from Leydig cells that are in direct contact with the
testicular microvasculature, and that the processes of synthesis and
secretion are intertwined. However, LH receptors were described
recently in testicular vascular endothelium, suggesting that LH has a
vasoactive function (5). For example, these receptors could mediate the
release of testicular hormones into plasma from the interstitial fluid.
Several years ago we found that the secretion of testosterone and
estradiol (6) as well as immunoactive inhibin (7) into spermatic vein
blood in men with varicocele-associated infertility occurred
simultaneously. In light of the proposed vasoactive role for LH, it is
possible that all testicular hormones in interstitial fluid are
released simultaneously into the circulation. If so, the relative
concentrations of these substances in spermatic vein plasma should be
proportional to their relative testicular concentration. The present
research was designed to pursue this hypothesis.
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Materials and Methods
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Seven men with varicocele-associated infertility volunteered for
the spermatic vein blood-sampling study conducted in 1985–1988
according to a protocol approved by the human investigation committee
of Montefiore Hospital and the University of Pittsburgh School of
Medicine. Mean plasma FSH, LH, and testosterone levels were normal for
each man. The internal spermatic vein was catheterized using a femoral
vein approach. The subjects were returned to a quiet room for the
sampling study, in which blood samples were drawn every 15 min for
4 h, and plasma was saved at -20 C for subsequent assay. Subjects
underwent balloon/coil occlusion of the spermatic vein as treatment for
varicocele immediately after the blood sampling. Testosterone secretion
results for six of the men were reported previously (6).
Diagnostic testicular biopsies were performed in a second group of 20
infertile men with varicocele who also had normal plasma levels of FSH,
LH, and testosterone. Testicular steroid levels in these patients were
reported previously (3) and are included in this report for the purpose
of comparison with spermatic vein plasma levels.
Determination of steroid concentrations in the spermatic vein
plasma and testis
Plasma samples were diluted 1:10 to 1:400 with
phosphate-buffered saline. After adding tracer amounts of
[3H]testosterone,
[3H]17-hydroxyprogesterone,
[3H]androstenedione, or
[3H]progesterone to monitor recovery,
samples were extracted with fresh diethyl ether. For androstenedione
and progesterone assays, extracts were purified on
Sephadex LH-20 columns using hexane-benzene-methanol (90:5:5,
vol/vol/vol), whereas testosterone and 17-hydroxyprogesterone were
assayed without chromatography. The recoveries of radiolabeled steroids
were at least 60%. All immunoassays used the dextran-coated charcoal
separation method. The antisera were purchased from Teikoku Hormone Co.
(Kawasaki, Japan) and have been described in detail previously (3). The
cross-reactivities of the antisera with testosterone were:
androstenedione antiserum, 5.4%; progesterone antiserum,
0.18%; and 17-hydroxyprogesterone antiserum, less than 0.04%. The
detection of nonlabeled testosterone in all assays was less than 0.1%.
All samples from a given study were analyzed in one immunoassay. The
within-assay coefficients of variation were less than 10%. Assays were
performed in samples stored frozen for no more than 2 yr.
Testicular tissue samples from men undergoing diagnostic testicular
biopsy for oligospermia were extracted immediately after surgical
excision using methanol (20 mL/20 mg testicular tissue). Tracer amounts
of [3H]testosterone, [3H]androstenedione,
[3H]progesterone, and
[3H]17-hydroxyprogesterone (
2000 cpm) were
dissolved in methanol and added to monitor losses during extraction and
chromatography. These steroid hormones were isolated by chromatography
on LH-20 minicolumns using benzene-methanol (90:10, vol/vol) and
measured using specific dextran-charcoal assays (3).
Data analysis
Data are presented as the mean ± SEM. Linear
regression was performed using SYSTAT for Windows, version 5 (Systat,
Evanston, IL).
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Results
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Mean spermatic vein steroid hormone levels over the course of the
4-h sampling study for each of the seven subjects are found in Table 1
. Testosterone was measured in highest
concentration, followed by 17-hydroxyprogesterone, androstenedione,
and progesterone, respectively. The results are rank
ordered for mean testosterone concentrations and reveal a substantial
range of mean values among subjects.
Figure 1 illustrates representative
profiles obtained from the measurement of spermatic vein plasma steroid
levels every 15 min for 4 h for two of the subjects. Serial
determinations of the concentrations of testosterone and its precursor
steroids revealed a pulsatile pattern of release into spermatic vein
plasma for each of the steroids studied. Notably, secretory pulses of
testosterone were coincident with those of the precursor steroids. By
contrast, the level of total plasma protein in the samples was quite
constant (not shown). The duration of the secretory events ranged from
15–90 min. The testosterone concentration between peak and trough
levels for a given subject varied by as much as 50-fold.
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Figure 1. Representative examples of testosterone,
17-hydroxyprogesterone, androstenedione, and
progesterone concentrations in spermatic vein plasma
samples obtained every 15 min for 4 h in two men with
varicocele-associated infertility.
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As shown in the example in Fig. 2, the
spermatic vein plasma level of testosterone was highly predictive of
the concentrations of each of the precursor steroids. For the group of
seven men, correlation analysis revealed a strong positive relationship
between the level of testosterone and the level of each of the
precursor steroids (Table 2).
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Table 2. Correlation coefficients relating testosterone in
spermatic vein blood to the levels of its 4 precursor
steroids
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For each subject, the sum of the values of the four steroids was
determined, and testosterone was found to account for 70 ± 1.9%
(mean ± SEM) of this value, followed by 24 ±
1.6% for 17-hydroxyprogesterone, 4.8 ± 1.8% for
androstenedione, and 1.1 ± 0.21% for progesterone.
The sum of the concentrations of testosterone,
17-hydroxyprogesterone, androstenedione, and
progesterone in a series of testicular biopsy specimens
from a second group of men with varicocele (3) was likewise calculated.
In that study the mean intratesticular concentration of testosterone
was 1700 ± 246 ng/g (range, 415-4814 ng/g), that of
17-hydroxyprogesterone was 540 ± 87 ng/g (range, 175-1784
ng/g), that of androstenedione was 99 ± 17 ng/g (range, 25–340
ng/g), and that of progesterone was 81 ± 9 ng/g
(range, 34–144 ng/g). Testosterone thus also accounted for 70% of
this sum. The pie chart in Fig. 3 shows
the striking similarity in the relative concentrations of testosterone
and its 4 precursor steroids in testis and spermatic
vein plasma.
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Figure 3. Relative concentrations of testosterone,
17-hydroxyprogesterone, androstenedione, and
progesterone in spermatic vein plasma in 7 men and in
testicular tissue from 20 men undergoing diagnostic testicular biopsy
for infertility.
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Discussion
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The data obtained in this study reveal that testosterone,
17-hydroxyprogesterone, androstenedione, and
progesterone are cosecreted from the human testis into
spermatic vein plasma. Moreover, the relative concentrations of these
four steroids in spermatic vein plasma and testis tissue are nearly
identical. In agreement with many previous studies, the most abundant
unconjugated steroid in spermatic vein plasma was testosterone,
followed by 17-hydroxyprogesterone, androstenedione, and
progesterone, respectively (8, 9, 10, 11, 12). The mean
concentrations of these four steroids in spermatic vein plasma in this
study are similar to results reported previously in men with varicocele
(8, 9, 10, 11) or inguinal hernias (12). Considerable variability in mean
values for a given hormone among subjects may be attributed partly to
the presence of collateral veins joining the testicular vein, with
dilution of testicular venous blood by splanchnic or renal blood as
well as to differences in Leydig cell function among subjects. No doubt
the wide range of individual sample values reported previously also
partly reflects peak and trough levels due to pulsatile release. This
is the first study to demonstrate that testosterone precursor steroids,
as well as testosterone, are released into spermatic vein plasma in a
pulsatile fashion. Our earlier finding of testosterone secretory
episodes of variable duration into spermatic vein plasma was recently
confirmed and was related to LH secretory episodes (13).
Small nonpolar molecules, such as steroid hormones, are presumed to
diffuse freely and rapidly across cell membranes, leading to the idea
that testosterone produced by Leydig cells enters the testicular
interstitium and diffuses across the capillary endothelium to produce a
secretory burst. If this mechanism is correct, testosterone
biosynthesis should be episodic, and testicular steroid concentrations
might be expected to fall to very low values between successive waves
of biosynthesis. Moreover, the secretion of the precursor steroids
might precede that of product testosterone. However, the maximum
10-fold variation in concentration for each testicular steroid measured
and the concordant secretion of testosterone and its 4
precursor steroids observed in spermatic vein plasma in this study
suggest, instead, that active membrane transport of gonadal steroids
out of the testis may occur, as proposed for testosterone entry into
cells coupled to sex hormone-binding globulin (14). Before rejecting
the diffusion hypothesis, however, a more frequent blood-sampling
protocol is needed to adequately evaluate secretory dynamics, and
studies of LH control of testosterone biosynthesis in real time in
humans must be performed.
The present findings allow for the alternate hypothesis that by acting
through specific receptors, LH alters the permeability of the
testicular vascular endothelium, allowing compounds in the interstitial
compartment to enter the circulation. The presence of binding sites for
hCG/LH in the vasculature of the reproductive tract was first described
in bovine corpora lutea using light microscopic autoradiography (15).
Subsequently, immunocytochemistry was used to demonstrate hCG receptors
in rat testicular blood vessels (16), and in situ perifusion
of the rat testicular vasculature with labeled hCG together with an
antiserum to the LH receptor revealed binding of hCG to receptors on
endothelial cell membranes (5). Blood vessel binding of hCG is organ
specific, as receptors are absent in the carotid artery (15) and in
vessels of the liver and kidney (17). Ghinea and Milgrom (18) have
proposed that receptors in endothelial cells mediate hormone
transcytosis (the passage of hormones from plasma into the interstitial
space), but how plasma hormones cross the endothelium remains
controversial (19).
Kinetic observations of hCG/LH-stimulated testosterone production
are consistent with the view that LH stimulates testosterone release
through a vascular mechanism. Testosterone levels increase in human
spermatic vein plasma simultaneous with the rise in plasma LH within 15
min of injecting GnRH into peripheral blood (20). On the other hand,
when human testicular biopsy specimens were stimulated with hCG,
testosterone production increased slightly at 30 min and increased
linearly for 6 h (21), suggesting, with the limitations of
extrapolating from in vitro results, that biosynthesis is
not episodic. Turner and Rhodes (22) examined the movement of labeled
LH from the vascular compartment into the interstitial fluid in rats
and found constant LH levels in interstitial fluid between 5–15 min
after injecting LH iv. From those data, they concluded that Leydig
cells are not exposed to LH pulses through the tubular interstitial
fluid and proposed that other factors initiate the release of
testosterone from the testes.
A role for the testicular vasculature in the process of testosterone
secretion has been proposed previously (23). When rats are treated with
hCG, testicular interstitial fluid volume rises because vascular
permeability increases (24). Moreover, testicular capillary
permeability declines after hypophysectomy (25). Total testicular blood
flow using the radioactive microsphere method is also increased by
hCG/LH (26). However, testicular permeability does not increase until
6–8 h after hCG administration (24, 26), and the increase in
testicular blood flow appears to follow the change in capillary
permeability (26, 27). Vasomotion (rhythmic variation in blood flow) is
inhibited by LH/hCG within 2–6 h (28). Thus, the causal relationship
of these delayed changes in the testicular vasculature to the rapid
release of testicular steroids from the LH-stimulated testis is
uncertain.
In conclusion, testosterone and its precursor steroids are released
simultaneously as pulses into spermatic vein plasma in proportion to
their concentrations within the testis. These data are consistent with
the presence of an active process that governs steroid hormone release
from the testicular interstitium. These observations were made in men
with varicocele-associated infertility, however, and their
applicability to normal men can only be assumed.
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Acknowledgments
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The authors acknowledge the expert technical assistance provided
by Ms. Joyce Szczepanski and Mr. Dushan Ghooray. We also thank Dr.
Matthew Hardy for his critical reading of the manuscript.
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Footnotes
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1 This work was supported in part by NIH Grant RO1-HD-19546 and
NIH/NCRR/GCRC Grant no. 5 M01 RR00056. A portion of these data was
published in Clin Res 36:392A, 1988. 
2 Current address: Department of Obstetrics and Gynecology, Showa
University School of Medicine, Tokyo, Japan.
Received September 16, 1998.
Revised December 4, 1998.
Accepted December 10, 1998.
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Abstract
Introduction
