Immune Endocrine interactions in the control
of Leydig cell function
An overview of research in theHales' laboratory (the first decade: 1990-2000)
Dale Buck Hales, PhD
copyright 2000 by dbh
contents
Chapter One, gonads, gametes and steroid
hormones
Chapter 2: Regulation of testosterone by immune factors
Chapter 3: in vivo analysis of immune regulation.
Figure 1: scanning electron
micrograph of a cross-section of the rat testis
Figure 2: The functional and anatomical compartmentalization
of the testis
Figure 3: testosterone biosynthetic pathway in
Leydig cells
Figure 4: putative model of StAR
mechanism of action.
Figure 5: mitochondrial vs. nuclear control of Leydig
cell steroidogenesis.
Figure 6: Antechinus Stuartii: victim of his own
testosterone or lessons from marsupial mice.
Chapter One, gonads, gametes and steroid hormones.
During the 1960’s careful analysis of the structure of the testes revealed an
unusual cellular association in the interstitial tissue. Testicular Leydig cells and
macrophages were shown to be intimately associated. This observation formed the basis of
my studies on immune-endocrine interactions in the testis and lead us to discover how
immune activities modulate testosterone production. Leydig cells are the cells in the
testis that make testosterone. In fact, they are essentially the only site of testosterone
production in the male. The Leydig cells are the major endocrine cells of the testis and
as such form the central cell in the endocrine "compartment". We think of the
gonads as being both functionally and anatomically compartmentalized into the
"gamete" and "endocrine" compartments. The gamete compartments in both
gonads is where the germ cells develop—the oocytes or eggs in the ovary, and the
spermatocytes or sperm in the testis. The endocrine compartment is where steroid hormones
are produced. Thus the functional compartmentalization is defined by the
compartment’s role, either as the site where the development of the gametes takes
place, or the site of steroid hormone production. Anatomically the compartments are
defined by the structures that form either the follicle where oocytes mature, or the
seminiferous tubule where spermatogenesis takes place. Each compartment is formed by a
distinct membranous structure that physically separates the gametes from the rest of the
gonad. The major difference between the ovarian compartment—the follicle, and the
seminiferous tubules, is that one follicle contains a single oocyte, whereas the tubules
are the site of continuous spermatogenesis. Millions of sperm are produced continuously
throughout the reproductive life of the male, where as oocytes are limited in number and
mature essentially as single entities. The endocrine compartments of each gonad are also
quite similar. The compartments reside in the interstitium of the gonad, the area between
the gamete compartments in the well vascularized tissue.
return to top
The Leydig cells in the interstitial tissue of the testis are uniquely positioned to
provide testosterone for to the seminiferous tubules to drive spermatogenesis, and to the
peripheral circulation to supply testosterone to the rest of the body to form and maintain
male secondary sexual structures and characteristics. The observation that Leydig cells
and macrophages are closely associated created speculation that these two cells are
functionally, as well as anatomically associated. The nature of the functional association
has been the subject of the work in my laboratory during the 1990s. Similar to the history
of Leydig cells, anatomical observation provided the basis for the elucidation of
function. Franz Leydig first described Leydig cells in the 1850s. He observed cells in the
testis with dense granules which were later shown to be lipid droplets. This morphological
feature predicted the function of Leydig cells. They convert cholesterol to testosterone,
and the lipid droplets are vesicles filled with cholesterol esters. These lipid droplets
provide cholesterol for testosterone production. Despite the observation of Leydig cells
in the testis, it wasn’t until the late 1950s and early 1960s that is was shown
unequivocally that they are the cells that produce testosterone. Work in the testis
followed studies in adrenal where similar investigations unravled the steps of steroid
hormone biosynthesis. Early studies determined that cholesterol is converted to
pregenenolone within the mitochondria and then pregnenolone is converted in the smooth
endoplasmic reticulum to the final steroid products. These studies also showed that the
first hormonally regulated step in steroid synthesis is the conversion of cholesterol to
pregnenolone.
During the 1970s the pathway of testosterone production was elucidated and shown to
comprise many of the same steps that result in adrenal steroid synthesis and was performed
by many of the same enzymes. The first enzyme in the pathway is cholesterol side-chain
cleavage enzyme referred to as P450scc. P450scc resides on the inner face of the inner
mitochondrial membrane. The next enzyme in the pathway is 3b-hydroxysteroid
dehydrogenase which is referred to as 3b-HSD. 3b-HSD converts pregnenolone to progesterone, the first biologically
active steroid hormone in the pathway. Progesterone is then converted to androstenedione
by the action of 17a-hydroxylase/c17-20 lyase enzyme, referred
to as P450c17. Androstenedione is the immediate precursor of testosterone. The final step
in the biosynthesis, conversion of androstenedione to testosterone is carried out by the
enzyme 17b-hydroxysteroid dehydrogenase, referred to as 17b-HSD. The same basic pathway is utilized for testosterone production
in all mammals, though the order in which the enzymes act may differ slightly. See
Figure 3: testosterone biosynthetic pathway in Leydig cells.
return to top
Another feature of steroid hormone synthesis common to the adrenals and gonads is the
control by pituitary tropic hormones. Adrenal glucocorticoid hormones such as cortisol are
under the control of adrenocorticotrophic hormone or ACTH. Ovarian and testicular steroid
hormone production is under the control of the gonadotropins, Luteinizing hormone and
follicle stimulating hormone, or LH and FSH, respectively. Testosterone production by
Leydig cells is under the control of LH. LH is secreted by the anterior pituitary and
travels via the blood stream to the testes where it binds to receptors on the surface of
the Leydig cells and stimulates testosterone production. LH is what is known as a
glycoprotein hormone and it can’t enter the cell. Instead it binds to receptors on
the outside surface of the cell and activates an intracellular second messenger system
inside the cell to provoke cellular responses. CyclicAMP, referred to as cAMP, is
LH’s second messenger in Leydig cells. LH actions in Leydig cells are signaled via
the production of cAMP which activates the enzyme protein kinase A and this enzyme,
protein kinase A carries out the work of cAMP and indirectly, LH to promote testosterone
production. The pathway from LH to testosterone production is also shown in
Figure 3.
Chapter 2: Regulation of testosterone by immune factors
The close association between macrohpages and Leydig cells suggest that these
interstitial cells in the testis may be functinoally related. We hypothesized that
macrophage secreted factors such as the proinflammatory cytokines interleukin-1 and tumor
necrosis factor may affect Leydig cell function, and Leydig cells are devoted to the
production of testosterone so we examined their effects on testosterone production. The
very first experiment we did revealed that IL-1 was a potent repressor of steroidogenic
enzyme gene expression in Leydig cells. The enzyme that converts progesterone to
androstendione, the immediate precurssor or testosterone is P450c17 and P450c17 is the
most sensitive enzyme in the biosynthetic pathway to cytokine inhibition. We have examined
TNF, IL-6 as well and they also inhibit Leydig cell steroidogenesis and P450c17
expression. It is intriguing to consider that P450c17 is susceptible to inhibition by
several different types of stimuli. Inflammatory mediators such as cytokines,
environmental pollutants such as dioxin, and testosterone itself inhibit the expression of
P450c17. To study the mechansims through which these different agents affect P450c17 we
have taken two approaches—signal transduction analyses and genetic analyses. Our
signal transduction studies indicate that TNF but not IL-1 activates protein kinase C and
PKC inhibits P450c17 transcription. Other agents such as arginine vasopressin and phorbol
esters which also activate PKC are similar and inhibit c17 expression. IL-6, IL-2,
interferon-g which do not activate PKC (similar to IL-1) also
inhibit c17. Likewise dioxin and testosterone neither activate PKC yet all of these
compounds that act through distinct signaling pathways all converge at one point in
testosterone biosynthesis and inhibit P450c17 transcription. Our genetic approach involves
cloning the gene and analyzing the 5’ flanking region that contains the genetic
control elements, the so called promoter. Promoter mapping has revealed a region of some
140 nucleotides that confers cAMP responsiveness to the gene. By selective mutation we
have altered regions of the promoter in a systematic fashion and found two control
regions, one repeated 4 times and another unique region. It appears that androgen and
dioxin act at one of these regions. We believe that these signaling pathways that inhibit
the gene work by blocking the cAMP factor from binding to gene. We think the cAMP factors
bind to the sequence that is repeated 4 times and to the region where the dioxin and
androgen receptors bind. We are trying to clone these cAMP factors now.
Chapter 3: in vivo analysis of immune regulation.
Our studies with isolated Leydig cells treated in vitro with cytokines, or in MA-10
tumor Leydig cells we use to study the promoter constructs demonstrated how sensitive c17
transcription is to cytokine inhibition. To study cytokine effects in vivo we inject mice
with endotoxin isolated from bacteria. The endotoxin we use is lipopolysaccharide which is
a component of the bacterial cell wall. LPS is what signals the immune system that
bacteria are present. Macrophages have LPS receptors that sense bacteria have invaded the
body. The macrohpages respond by producing cytokines and the cytokines produce all of the
symptoms that accompany bacterial infection such as fever, edema, etc. Instead of actually
injecting mice with bacteria we use LPS which evokes much the same response. When we did
this we observed that c17 expression was indeed inhibited as we expected. We first looked
24 hours after injection and saw that c17 was completely gone! Careful studies showed that
LPS causes testicular macrophages to produce cytokines but that Leydig cells themselves
are not sensitive to LPS directly. We were very excited and pleased to observe that our in
vitro studies had predicted what happens in the animal.
An important consideration was how fast steroidogenesis was affected following LPS
injection. It turns out that testosterone levels plummet soon after injecting LPS, yet the
steroidogenic enzyme RNAs do not decrease for hours. This conundrum became the focus of
our studies. What mechanism could account for such a rapid decrease in testosterone even
though the enzymes appear to be unaltered? It was about this time that the cholesterol
transfer pathway in steroidogenesis was elucidated. The steroidogenic acute regulatory
protein, a.k.a. StAR was identified and reagents became available to examine StAR
expression. While P450c17 is unaltered at 2 hours, it appeared that StAR protein was
markedly reduced, concomitant with the decrease in testosterone. This compelling
observation suggested that some inflammatory signal shuts of testosterone synthesis by
arresting the flow of substrate into the pathway. It makes sense, actually, if you want to
stop the flow of water out of a hose, you turn it off at the spigot. The problem we
encounterd though, was that we were unable to reproduce the acute and immediate inhibition
of StAR and testosterone production in vitro in our cell culture models. We treated the
cells with a host of inflammatory mediators, the very agents that block c17 transcription
had no acute inhibitory effects. We did many laborious studies looking at the effects of
LPS in vivo, injecting animals, removing their testes and isolating then analyzing the
Leydig cells. From these studies we learned much, but were limited in our mechanistic
analyses. One thing we did observe was the accumulation of the 37 kDa precrussor of StAR.
StAR is a nuclear encoded gene product that is translated in the cytoplasm, then
"processed" from a 37 kDa form to the 30 kDa mature form. It undergoes
processing while it is being translocated from cytoplasm to the inner mitochondrial
membrane. The observed accumulation of the 37 kDa form in suggested that StAR was not
being processed. For sometime we believed that the inability of StAR to be processed was
why cholesterol couldn’t be transferred. Without being able to model this in vitro,
though, it was a difficult hypothesis to test.
 |
Figure 4: putative model of StAR
mechanism of action. C' terminal region is critical for
cholesterol transfer and may associate with as yet unidentified outer
mitochondrial membrane component resulting in the trasient formation of a
cholesterol "pore" or contact site that enables cholesterol to
transit the innermitochondrial space and enter the matrix where it is
immediately converted to pregnenolone via the action of the P450scc enzyme
complex, then in turn converted to progesterone via the action of 3b-HSD. |
return to top
Much has been learned about StAR since it was first cloned some 5 years ago now. The
actual mechanism through which it facilitates cholesterol transfer is as yet undetermined.
We now know that StAR does not require processing to participate in cholesterol transfer.
We believe that the translocation and processing step is the way StAR is turned off. Since
StAR is the acute regulatory protein, it has to be produced immediately to start
testosterone production, but it also has to be quickly shut off, so that testosterone is
produced on demand, not continuously. By translocating into the mitochondria StAR somehow
enables cholesterol to get into the mitochondria, and then once it is inside, it is
rendered inactive. The next pulse of LH from the pituitary will stimulate a new bout of
StAR synthesis, cholesterol transfer, and then cessation of cholesterol movement. The
transfer of cholesterol itself only requires that StAR associates transiently with the
outer mitochondrial membrane and this evidently triggers the opening of a cholesterol
"pore" which allows cholesterol to get into the inner mitochondria. This would
indicate, then, that processing per se is not required for cholesterol transport. In light
of these findings, our hypothesis that an LPS generated signal interfers with StAR
processing causing the block to cholesterol transfer was seriously challenged. None the
less, our obsevation that StAR processing was blocked coincident with the cessation of
cholesterol transfer had to mean something!.
Mitochondria are the site of cellular respiration where ATP, the cells energy currency
is produced. ATP synthesis takes place within the inner mitochondria by converting energy
derived from metabolism. Every electron’s worth of energy is squeezed out of the
nutrients during respiration. The flow of electrons down a gradient of electron transport
proteins eeks all of the possible energy out of the nutrient molecule. During this process
the electrons are converted to protons and shuttled out of the inner mitochondria into the
space between the inner and outer mitochondrial membranes. This accumulation of protons in
the space generates the force that drives ATP synthesis. Nature seek equlibrium. The
uneven distribution of protons between the inner mitchondrial membrane and space causes
the generation of an electronegative potential inside the inner mitochondria. Protons are
positively charged. Each one that gets pumped out during electron transport causes the
inside to become more negative. The energy from this proton gradient is harnased by the
enzyme that synthesizes ATP. ATP is produced by phosphorylating ADP (going from 2
phosphates to 3 phosphates attached to the adenine nucleside). As nutrient molecules are
metabolized, they are oxidized, analogous to being burned up. Thus ATP synthesis is
produced by oxidative-phosphorylation, and results from and is dependent on the protein
gradient within the mitchondria. This gradient is referred to as DYm.
Agents that poke holes in mitochondria and disrupt DYm
block ATP synthesis.
It turns out that pertubation of DYm also
inhibits cholesterol transfer—and StAR processing. One agent that pokes holes in
mitochondria, so to speak, is carbonyl cyanide chlorophehylhydrzone, or cccp. CCCP is a
"protonophore" which means it creates a pore for protons and allows them to
equilibrate across the mtiochondrial membranes. This disipates DYm.
Our friend Yossi Orly showed that CCCP not only disrupst DYm,
it also blocks StAR processing and cholesterol transfer. When we saw his data we
reformulated out hypothesis and proposed that LPS causes the disruption of DYm with consequent blockade to cholesterol transfer and
perturbation of StAR processing. Thus seeing the accumulation of the 37 kDa form of StAR
did mean something—that the mitochondria were perturbed and this is what evidently
blocks cholesterol transfer. The discovery that agents which dissipate DYm
mimic LPS action was a huge breakthrough for us. Now we can effectively model the acute
inhibitory phase of the LPS response in vitro and conduct detailed mechanistic analyses.
However, one essential piece of data was required. The demonstration that LPS in fact does
disipate DYm in vivo.
To analyze mitochondrial polarity electrochemical dependent vital dyes are used. The one
we employ is tetramethyl rhodamine ethylester or TMRE. IF cells have an intact DYm then TMRE fluoresces and if they don’t, it wont.
This provides a very sensitive and unambiguous assessment of the state of mitochondria in
cells. We first incubated MA-10 cells with CCCP and then stained them with TMRE to work
out the method. Then we isolated Leydig cells from LPS injected mice and stained them with
TMRE. Voila! DYm was indeed disrupted. WE now have
confidence that agents which disrupt the mitochondria also block cholesterol transfer and
StAR processing, and if StAR can’t be processed, then it gets degraded. So, our
observation of the 37 kDa form of StAR accumulating in cells from LPS injected mice turned
out to be the key to unraveling the mechanism of LPS action.
return to top
LPS itself has no effect on Leydig cells since they lack the LPS receptor. And logic
dictates that LPS does not stimulate the production of CCCP within the cell, so our next
question becomes, what signal does LPS generate that perturbs the mitochondria? We know
that LPS stimulates macrophages to produce cytokines, this is a process that takes several
hours to be fully manifest. Another well described action of macrophages is to produce
reactive oxygen in response to LPS. When macrophages sense bacteria they "know"
they need to destroy them so they produce activated forms of oxygen that are highly
reactive and destroy the bacteria. These reactive oxygen species work over a short
distance and attack lipids, proteins and nucleic acids and destroy them. The cell has
natural antioxidant protective mechanisms to minimize the damage to innocent bystander
cells. Antioxidant vitamins (A, C, E) and other agents such as glutathione soak up the
radicals and neutralize them. However, rampant and uncontrolled reactive oxygen molecules
overwhelm the cellular antioxidants and damage cells. Mitochondria are well known targets
for reactive oxygen. So, considering the proximity of macrophages to Leydig cells, that
they produce reactive oxygen in response to LPS suggested the hypothesis that LPS
stimulated the production of reactive oxygen molecules and these agents damaged the Leydig
cell mitochondria. To test this we put hydrogen peroxide on Leydig cells and indeed
determined that cholesterol transfer and StAR processing were blocked. We are now looking
at TMRE fluorescence in hydrogen peroxide treated cells to determine if DYm is dissipated—we anticipate that it will be.
One of he consequences of depolarizing mitochondria is the initiation of the cell death
program known as apoptosis. We speculate that LPS may cause Leydig cells to under
apoptosis and this would account for the sustained perturbation of testosterone we have
observed. Our future studies will include a detailed analysis of apoptosis in Leydig cells
from LPS injected mice—as well as in MA-10 cells treated with hydrogen peroxide.
return to top
Chapter 4:significance of immune modulation of testosterone
What is the advantage of immune inhibition of testosterone ? Why would the testis
evolve to have this elaborate inhibitory mechanism in place? There are several potential
reasons. If your are sick you should conserve your energy and not devote it to
reproduction, but channel it to healing. If you are sick it is to the advantage of the
population that you not reproduce—you would be unable to provide for your offspring
or you might be genetically susceptible to illness and would propagate this defect if you
reproduced. Testosterone not only drives the libido, but it is the hormone of aggression.
Testosterone behavior is the opposite from sickness behavior, so if you are sick you
shouldn’t fight. This would be an immediate benefit to this inhibitory action. Also,
one could argue that testosterone is immunosuppressive and for the animal to wage the
maximal possible immune response it would be to its advantage to shut off testosterone at
its source. The incidence of autoimmunity in women compared to men supports the immune
suppressive theory of testosterone action. It is also possible that there is no
evolutionary advantage to the inhibitory interaction between macrophages and Leydig cells,
but that it is a pathophysiological consequence of inflammatory disease.
Figure 6: Antechinus Stuartii
victim of his own testosterone or lessons from marsupial mice. Nature
provides an example that is consistent with the hypothesis: that in order
for the animal to mount an effective immune response, products of the
immune reaction, cytokines, inhibit the production of immunosuppresive
androgens. Male marsupial
mice, Antechinus stuartii, are overtly preoccupied with copulation and die
abruptly at the conclusion of the mating season. |
During
the mating season, males have dramatically elevated serum testosterone
levels.
In
contrast, female A. Stuartii are
longer lived and survive several mating seasons. To determine the
mechanism behind the difference in longevity between males and females,
autopsies of males revealed a variety of disease states, all associated
with suppression of immune and inflammatory responses.
It was observed that castrated males survived in the field well
beyond the period of natural mortality.
Males who were captured and raised in a pathogen-free environment
lived up to three years, equivalent to the life span of females. These
observations support the hypothesis that androgens are immunosuppressive.
In addition to direct inhibitory effects on the immune system,
androgens inhibit corticosteroid binding globulin synthesis which results
in an increase in free circulating glucocorticoids causing a further
suppression of the immune response. Therefore,
inhibition (or removal) of androgens allows the animal to mount an
effective immune response. |
return to top
suggested further reading
- Hales D.B., Diemer T., Held Hales K. "Review: The Role of
Cytokines in Testicular Function" Endocrine 10:201-217 ( 1999)
- Hales,
D.B."Cytokines
and Testicular Function" in Cytokines
and Reproduction (Hill, J.A. ed.) John Wiley & Sons, NY, NY. pp.
17-42 ( 2000)
- Hales, D.B. "Macrophage-Leydig Cell Interactions: An
Overview" in The Leydig Cell (Payne, A.H., Hardy, M.P., Russell,
L.D. eds.).
Cache River Press, Vienna, IL, pp. 451-466 (1996)
DB Hales' PowerPoint presentations
that cover this topic:
Buck's Faculty Page||Hales Lab Page||Physiology & Biophysics Homepage ||UIC Home Page ||Buck's
Own Home Page
Email to Buck
